JP2023103347A - Potentiation of lytic protein anti-bacterial activity with blood component, and methods and uses thereof - Google Patents

Abstract

To provide methods, assays, compositions, formulations and constructs, particularly lytic peptide constructs, which relate to and are based on the activity and use of blood components, particularly serum albumin and lysozyme, and their activity and use to enhance or synergize with the bacterial killing effect of anti-bacterial lytic proteins and peptides.SOLUTION: The invention provides a composition or combination for enhanced or synergistic killing of Gram-positive bacteria. The composition or combination comprises an isolated lysin polypeptide having an SH3 type binding domain, and one or more blood component proteins selected from serum albumin and lysozyme, or a peptide or fragments thereof, where the peptide or fragments thereof demonstrate activity of the full-length albumin or lysozyme protein with regard to lysin polypeptide activity enhancing effects.SELECTED DRAWING: Figure 1A

Description

The present invention relates generally to the activity and use of blood components, particularly serum albumin and lysozyme, and antibacterial lytic proteins and peptides, to enhance or synergize with the bactericidal effect thereof. The invention further relates to lytic peptide constructs, formulations, assays and methods based on blood components and/or peptides or proteins thereof.

Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharides. The Gram-positive cell wall is 20-80 nm thick and appears as a broad dense wall composed of numerous interconnected layers of peptidoglycan. Between 60% and 90% of the Gram-positive cell wall is peptidoglycan, which gives the cell shape, rigid structure and resistance to osmotic shock. The cell wall does not exclude Gram-staining crystal violet, which stains the cell purple, and is therefore "Gram-positive." Gram-positive bacteria include, but are not limited to, Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus
aureus) and Enterococcus faecalis. Spore-forming Bacillus spp. cause anthrax and gastroenteritis. Spore-forming Clostridium spp. are involved in botulism, tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium spp. cause diphtheria and Listeria spp. cause meningitis.

Antimicrobial agents that inhibit cell wall synthesis, such as penicillins and cephalosporins, interfere with the interpeptide linkages of peptidoglycan, embrittlement of the cell walls of both Gram-positive and Gram-negative bacteria. Gram-positive bacteria are more susceptible to these antibiotics because their peptidoglycan is exposed. Advantageously, eukaryotic cells lack cell walls and are less susceptible to these drugs or other cell wall agents.

The development of drug resistance, especially antibiotic-resistant bacteria, is a major problem in medicine as more and more antibiotics are used for a wide range of diseases and other conditions. Newer antimicrobial therapeutic approaches include enzyme-based antibiotics (“enzybiotics”) such as bacteriophage lysins. Phages use these lysins to digest the cell walls of their bacterial hosts, releasing viral progeny by hypotonic lysis. The high lethal activity of lysins against Gram-positive pathogens makes them attractive candidates for development as therapeutic agents (1, 2). Bacteriophage lysins were first proposed for eradication of nasopharyngeal carriage of pathogenic streptococci (Non-Patent Document 3, Non-Patent Document 2).

Bacteriophage lytic enzymes have been established as useful in the assessment and specific treatment of various types of infections in subjects by various routes of administration. For example, US Pat. No. 6,300,000 (Fischetti et al.) relates to rapid detection of group A streptococci in clinical samples via enzymatic digestion with semi-purified group C streptococcal phage-associated lysinases. This enzymatic research laid the groundwork for further research leading to cures for the disease. The Fischetti and Loomis patents (US Pat. Nos. 4,530,302, 4,500,002) disclose the use of lysin enzymes produced by Group C Streptococcus bacteria infected with C1 bacteriophage. US Pat. No. 5,300,000 (Fischetti and Loomis) discloses compositions against skin infections using lytic enzymes in a carrier suitable for topical application to skin tissue. US Pat. No. 5,300,000 (Fischetti and Loomis) discloses a method of treating bacterial infections of the gastrointestinal tract comprising administering lytic enzymes specific for the infecting bacteria. Fischetti and Loomis 7 discloses methods and compositions for the treatment of bacterial infections by parenteral introduction (intramuscular, subcutaneous or intravenous) of at least one lytic enzyme produced by a bacterium infected with a bacteriophage specific for that bacterium and a carrier suitable for delivery of the lytic enzyme to a patient.

WO 2005/020000 and WO 2005/020000 and WO 2005/020011 provide different phage-associated lytic enzymes, PlyG, γ and W, and PlyPH, respectively, useful as antimicrobial agents for the treatment or reduction of Bacillus anthracis infection. US Pat. No. 6,200,000 describes a Pal lytic enzyme against Streptococcus pneumoniae. Enterococcus genus (Enterococcus faecalis including vancomycin-resistant strains and Enterococcus faecium (E.
faecium)), in particular PlyV12, are described in US Pat. US Pat. No. 6,200,000 describes a mutant PlyGBS lysin that is highly effective in killing group B streptococci. A chimeric lysin, designated ClyS, with activity against Staphylococcus aureus, including Staphylococcus aureus, is detailed in US Pat. A PlySs2 lysin isolated from Streptococcus suis and effective in killing Streptococcus, Staphylococcus, Enterococcus and Listeria strains is described in US Pat.

PlySs2 lysin (also referred to herein as CF-301, CF301, PlySs2/CF-301, PlySs2(CF-301)) is the first lysin to enter and complete an FDA-approved Phase I clinical trial. PlySs2 lysins are described in US Pat. PlySs2 (CF-301) lysin can be combined with standard of care antibiotics (including but not limited to vancomycin or daptomycin) for the treatment of bloodstream infections, including endocarditis, caused by methicillin-sensitive and resistant Staphylococcus aureus.

In support of clinical trials, in vitro antibiotic susceptibility testing (AST) is used to evaluate and standardize antimicrobial agent(s). Although Broth microdilution (BMD) can be used to test the activity of lysins such as PlySs2 (CF-301) against S. aureus isolates, the standard method (CLSI methodology) is not a reliable assay and presents various problems when applied to lytic polypeptides such as PlySs2 (CF-301). PlySs2 (CF-301) is more effective in human blood, serum and plasma than artificial media. Understanding the enhanced activity and functionality of lysins such as PlySs2 (CF-301) in human blood, serum and plasma can lead to new and useful improved antimicrobial approaches, methods and therapeutics.

Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.

U.S. Pat. No. 5,604,109 U.S. Pat. No. 5,985,271 U.S. Pat. No. 6,017,528 U.S. Patent No. 6,056,955 U.S. Pat. No. 6,248,324 U.S. Pat. No. 6,254,866 U.S. Pat. No. 6,264,945 U.S. Pat. No. 7,402,309 U.S. Pat. No. 8,580,553 U.S. Patent No. 7,638,600 U.S. Pat. No. 8,389,469 U.S. Patent No. 7,569,223 U.S. Pat. No. 7,582,291 U.S. Pat. No. 8,105,585 WO2010/002959 U.S. Patent No. 8,840,900 WO2012/145630 U.S. Pat. No. 9,034,322 International application PCT/US2012/34456

Fischetti, V.A. (2008) Curr Opinion Microbiol 11:393-400 Nelson, D.L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112 Loeffler, J. M. et al (2001) Science 294: 2170-2172 Gilmer DB et al (2013) Antimicrob Agents Chemother Epub 2013 April 9 [PMID 23571534]

In a general aspect, the present invention relates to the identification and characterization of additional novel abilities of lysins, particularly lysin(s) polypeptides having SH3-type binding domains, including PlySs2 (CF-301) lysins, to interact with potential antimicrobial agents in human blood and enhance bacteriolysis. The present invention relates to the unique properties of lysin polypeptides, particularly lysin(s) polypeptides with SH3-type binding domains, including PlySs2 (CF-301) lysins, that exhibit synergy with and effect activation of blood components that have no or limited intrinsic antibacterial activity, particularly antistaphylococcal activity of their own, thereby allowing for maximal bactericidal activity.

The present application relates to activity-enhancing effects of blood component proteins, particularly serum albumin and lysozyme, particularly human serum albumin and human lysozyme, and antibacterial lytic peptides, particularly lysin. In one aspect, the serum albumin is selected from human serum albumin, rabbit serum albumin, dog serum albumin and horse serum albumin. Serum albumin, particularly human serum albumin, includes lysin polypeptides, particularly lysin polypeptides having SH-3 type binding domains, such as PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, particularly PlySs2 (CF-30 1) enhancing or otherwise enhancing the antibacterial activity of a lysin polypeptide selected from lysins; In one aspect, the lysozyme is human lysozyme. Lysozyme, particularly human lysozyme, serves to enhance or otherwise enhance the antibacterial activity of lysin polypeptides, particularly PlySs2 (CF-301) lysins. In one aspect, the combination of lysin polypeptides, particularly PlySs2 (CF-301) lysin polypeptides, and human lysozyme act synergistically to kill Gram-positive bacteria.

In one aspect of the invention, combinations of lysin polypeptide(s) and one or more blood component proteins are provided. In one aspect of the invention there is provided a combination of lysin polypeptide(s) and one or more blood component proteins selected from serum albumin and lysozyme. In one aspect, a combination of a lysin polypeptide having an SH3-type binding domain and one or more blood component proteins selected from serum albumin and lysozyme is provided. In particular embodiments, a lysin polypeptide having a SH3-type binding domain and selected from: PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, or potent variants thereof capable of binding to Gram-positive bacteria, including Staphylococcus spp. Compositions or combinations are provided comprising one or more blood component proteins selected from zozyme. In certain aspects, compositions or combinations are provided comprising a lysin polypeptide selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, or effective variants thereof capable of binding Gram-positive bacteria, including Staphylococci, and one or more blood component proteins selected from serum albumin and lysozyme, having a SH3-type binding domain.

In one aspect of the invention, synergistic combinations of lysin polypeptide(s) and one or more blood component proteins are provided, wherein the one or more blood components have no or limited endogenous antibacterial activity in the absence of the lysin polypeptide(s). In one aspect of the invention, the combination of the lysin polypeptide(s) and one or more blood component proteins has synergistic killing activity against Gram-positive bacteria, particularly Staphylococcus bacteria. In one aspect of the invention, synergistic combinations of lysin polypeptide(s) and one or more blood component proteins selected from serum albumin and lysozyme are provided. In one aspect, a synergistic combination of a lysin polypeptide having an SH3-type binding domain and one or more blood component proteins selected from serum albumin and lysozyme is provided. In certain aspects, compositions or synergistic combinations are provided comprising a lysin polypeptide having a SH3-type binding domain and selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, or effective variants thereof capable of binding Gram-positive bacteria, including Staphylococci, and one or more blood component proteins selected from serum albumin and lysozyme. In one aspect, the composition or combination further comprises one or more serum fatty acids, eg selected from oleate and palmitate.

In one aspect of the invention, lysozyme, particularly human lysozyme, is effective against Staphylococcus aureus bacteria when combined with the lysin polypeptide PlySs2 (CF-301). In one aspect of the invention, lysozyme, particularly human lysozyme, is effective against Staphylococcus aureus bacteria when combined with a lysin polypeptide or otherwise in the presence of a lysin polypeptide. In one aspect, the Staphylococcus aureus bacterium is susceptible to lysozyme in other forms, particularly human lysozyme, when combined with or in the presence of the lysin polypeptide PlySs2 (CF-301).

In one aspect of the invention, lysozyme, particularly human lysozyme, is effective against Staphylococcus aureus bacteria when combined with a lysin polypeptide having an SH3-type binding domain or otherwise in the presence of a lysin polypeptide having an SH3-type binding domain. In one aspect of the invention, serum albumin, particularly human serum albumin, enhances or otherwise enhances the antibacterial activity of lysin polypeptides having SH3-type binding domains.

In certain aspects of the invention, the lysin polypeptide having an SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, or potent variants thereof capable of binding Gram-positive bacteria, including Staphylococci. In certain aspects of the invention, the lysin polypeptides having SH3-type binding domains are PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin, AL
E-1 lysins or potent variants thereof capable of binding Gram-positive bacteria, including Staphylococci.

The present application relates to improved blood component-based methods and assays for determining the minimum inhibitory concentration of peptides, particularly antibacterially effective peptides, particularly lytic peptides, and assessing antibacterial killing effectiveness. The present application provides methods and assays for determining the minimum inhibitory concentration and assessing the antimicrobial killing efficacy of peptides, particularly antimicrobially effective peptides, particularly lytic peptides, to which serum albumin and/or lysozyme are added to accurately determine and/or predict the antimicrobial killing efficacy and/or the minimum inhibitory concentration of lytic peptides, including PlySs2 (CF-301), in animals or in vivo, particularly in humans. In one aspect, human lysozyme is added. In one aspect, human serum albumin is added. In one aspect, serum albumin is added which is derived from or corresponds in sequence to human, rabbit, dog or horse serum albumin. In one aspect, both serum albumin and lysozyme are added.

The present invention relates to a system or assay for determining the MIC of antimicrobial peptides, particularly lytic peptides, or for evaluating or quantifying antimicrobial activity and/or efficacy, wherein the assay is performed using an assay solution, broth or medium supplemented with serum albumin. The present invention relates to a system or assay for determining the MIC of antimicrobial peptides, particularly lytic peptides, or for evaluating or quantifying antimicrobial activity and/or efficacy, wherein the assay is performed using an assay solution, broth or medium supplemented with lysozyme. The present invention relates to a system or assay for determining the MIC of antimicrobial peptides, particularly lytic peptides, or assessing or quantifying antimicrobial activity and/or efficacy, wherein the assay is performed using assay solutions, broths or media supplemented with serum albumin and lysozyme. In one aspect of the invention, assays are provided for determining the bactericidal efficacy of antimicrobial peptides that accurately reflect the bactericidal efficacy of antimicrobial peptides, such as lytic peptides or lysins, in mammals or patients, particularly humans.

In one aspect, the lysozyme is human lysozyme. In one aspect, the serum albumin is human, rabbit, canine, equine, rat or bovine (bovine/bovine subfamily) serum albumin or corresponds in amino acid sequence to human, rabbit, canine, equine, rat or calf (bovine/bovine subfamily) serum albumin. In one aspect, the serum albumin is human, rabbit, dog, or horse serum albumin, or corresponds in amino acid sequence to human, rabbit, dog, or horse serum albumin. In one aspect, the serum albumin is homologous to naturally occurring human, rabbit, canine or equine serum albumin and differs in amino acid sequence by one or more amino acid sequences from naturally occurring human, rabbit, canine or equine serum albumin. Serum albumin or lysozyme used in the present invention may be purified or recombinant. Serum albumin or lysozyme may be a full-length protein, or a peptide or fragment thereof, wherein the peptide or fragment exhibits the activity of the full-length protein with respect to lysin polypeptide activity-enhancing effects and/or with respect to lysin polypeptide binding ability. Serum albumin or lysozyme may be a full-length protein, or a peptide or fragment thereof, wherein the peptide or fragment exhibits increased activity relative to the full-length protein with respect to lysin polypeptide activity-enhancing effects and/or with respect to lysin polypeptide binding capacity. In one aspect, the amino acid sequence of serum albumin, or a peptide or fragment thereof, differs from the native sequence. In one aspect, the amino acid sequence of serum albumin, or a peptide or fragment thereof, has greater enhancing activity or improved lysin polypeptide binding activity than the native sequence.

According to the present invention, there is provided a method of determining the bacterial killing activity of an antimicrobial peptide, such as a lytic polypeptide or a lysin, wherein the killing activity accurately mimics the bacterial killing of said antimicrobial peptide in humans, comprising evaluating the antimicrobial peptide in a broth, assay medium or solution supplemented with serum albumin isolated from or corresponding to human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or effective peptides or fragments thereof. In one aspect of the invention, lytic polypeptides or lysins are evaluated against susceptible bacteria in solutions, media or broths supplemented with human serum albumin, horse serum albumin, dog serum albumin or rabbit serum albumin. In one aspect, a reducing agent is additionally added to the broth, assay medium or solution. In one aspect, the reducing agent is DL-dithiothreitol (DTT). In one aspect, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP). In one aspect, 0.5 mM DL-dithiothreitol (DTT) is added to the solution, medium or broth.

According to the present invention, improved and improved microdilution (BMD) methods and assays for testing peptides, particularly antimicrobially effective peptides, particularly lytic or lysin peptides, are provided. In one aspect of the invention there is provided an improved BMD using a broth or medium for evaluation, wherein the broth or medium is supplemented with serum albumin, particularly human serum albumin, horse serum albumin, dog serum albumin or rabbit serum albumin. In one aspect of the invention there is provided an improved BMD using broth or medium for evaluation, wherein the broth or medium is supplemented with lysozyme, particularly human lysozyme. In one aspect of the invention there is provided an improved BMD using a broth or medium for evaluation, wherein the broth or medium is supplemented with serum albumin, particularly human serum albumin, horse serum albumin, dog serum albumin or rabbit serum albumin, and lysozyme, particularly human lysozyme.

The amount of serum albumin added can be determined by comparison with human serum. In one aspect, the amount of serum albumin added is equivalent to the amount normally present in a sample of human blood or serum.

The amount of lysozyme added can be determined by comparison with the normal amount of lysozyme present in human samples. In one aspect, the amount of lysozyme added is comparable to the amount normally present in a sample of human blood or serum.

In one aspect, the amount of reducing agent is between 0.1 mM and 10 mM. In one aspect, the amount of reducing agent is between 0.1 mM and 5 mM. In one aspect, the amount of reducing agent is between 0.1 mM and 2 mM. In one aspect, the amount of reducing agent is between 0.1 mM and 1 mM. In one aspect, the amount of reducing agent is between 0.1 mM and 0.9 mM. In one aspect, the amount of reducing agent is between 0.1 mM and 0.6 mM. In one aspect, the amount of reducing agent is between 0.2 mM and 0.6 mM. In one aspect, the amount of reducing agent is between 0.3 mM and 0.6 mM. In one aspect, the amount of reducing agent is between 0.4 mM and 0.6 mM. In one aspect, the amount of reducing agent is about 0.5 mM. In one aspect, the amount of reducing agent is between 0.25 mM and 1 mM. In one aspect, the amount of reducing agent is less than 1 mM.

In one embodiment, the assays and methods of the invention are used to assess and analyze lytic polypeptides. In one aspect of the invention, the BMD method with additive(s) is used to determine the bactericidal efficacy of lytic polypeptides active against streptococcal bacteria. In one aspect of the invention, the BMD method with additive(s) is used to determine the bactericidal efficacy of lytic polypeptides active against streptococcal and staphylococcal bacteria. In one aspect of the invention, the BMD method with additive(s) is used for MIC testing of lytic polypeptides active against streptococcal bacteria. In one aspect of the invention, the BMD method with additive(s) is used for MIC testing of lytic polypeptides active against Staphylococcus bacteria. In one aspect of the invention, the BMD method with additive(s) is used for MIC testing of lytic polypeptides active against streptococcal and staphylococcal bacteria. In one aspect of the invention, the additive(s)
is used for MIC testing of lytic polypeptides active against Enterococcus. In one aspect of the invention, the BMD method with additive(s) is used for MIC testing of lytic polypeptides against Gram-positive bacteria. In one aspect of the invention, the BMD method with additive(s) is used for MIC testing of lytic polypeptides against two or more species of Gram-positive bacteria. Gram-positive bacteria can be selected from the genera Streptococcus, Staphylococcus, Enterococcus and Listeria. Gram-positive bacteria can be antibiotic-resistant or antibiotic-sensitive bacteria.

According to the present invention novel, such as effective antibacterial compositions, comprising a lysin polypeptide, in particular a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, in particular human serum albumin, rabbit serum albumin, canine serum albumin, horse serum albumin, rat serum albumin, bovine (bovine/bovine subfamily) serum albumin, or active or binding peptides or fragments thereof, and/or lysozyme, in particular human lysozyme, or active peptides or fragments thereof. or improved formulations are provided. According to one aspect of the present invention there is provided a new or improved formulation, such as an effective antibacterial composition, comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or active or binding peptides or fragments thereof, and/or lysozyme, particularly human lysozyme, or active peptides or fragments thereof. In one aspect, a lysin polypeptide, particularly PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin, ALE-1 lysin, or capable of binding to and/or killing Gram-positive bacteria, particularly Staphylococcus or Streptococcus or Enterococcus bacteria. Effective antimicrobial compositions are provided comprising a lysin polypeptide selected from effective variants thereof and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin, or active or binding peptides or fragments thereof, or lysozyme, particularly human lysozyme, or active peptides or fragments thereof. In another aspect, lysin polypeptides, particularly PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin, ALE-1 lysin, or Gram-positive bacteria, particularly Staphylococcus or Streptococcus or Enterococcus, are capable of binding to and/or killing the same. An effective antimicrobial composition is provided comprising a lysin polypeptide selected from possible effective variants thereof and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or effective or binding peptides or fragments thereof, or lysozyme, particularly human lysozyme, or effective peptides or fragments thereof. In one aspect, an antimicrobial composition is provided comprising PlySs2 (CF-301) lysin, or a variant thereof effective in binding to and/or killing Staphylococcus and Streptococcus bacteria, and serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin. In one aspect, an antimicrobial composition is provided comprising PlySs2 (CF-301) lysin, or a variant thereof effective in binding to and/or killing Staphylococcus and Streptococcus bacteria, and lysozyme, particularly human lysozyme. In one aspect, the formulation is a topical or inhaled formulation. In one aspect, the composition is formulated for efficacy against skin infections or against pulmonary or oral infections.

In one aspect, a composition for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin, or active or binding peptides or fragments thereof. A topical formulation is provided. In another aspect, topical formulations of compositions for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, are provided comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or active or binding peptides or fragments thereof. In one aspect, topical formulations of compositions for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, are provided comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and a lysozyme, particularly human lysozyme, or an active peptide or fragment thereof. In one aspect, a composition for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin, or active or binding peptides or fragments thereof. Formulations for inhalation or orally administered are provided. In another aspect, there is provided an inhaled or orally administered formulation of a composition for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and one or more serum albumins, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or active or binding peptides or fragments thereof. In one aspect, there is provided an inhaled or orally administered formulation of a composition for treating Gram-positive bacterial skin infections, particularly Staphylococcal or Streptococcal skin infections, comprising a lysin polypeptide, particularly a lysin polypeptide having an SH3-type binding domain, and a lysozyme, particularly human lysozyme, or an active peptide or fragment thereof. In certain aspects, the topical or inhaled formulation is for the treatment of Staphylococcus aureus infections. In certain aspects, the topical or inhaled formulation is for the treatment of antibiotic-resistant Staphylococcus aureus infections. In one aspect, the lysin polypeptide having a SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. In one aspect, the lysin polypeptide having an SH3-type binding domain is a PlySs2 lysin or effective variant thereof.

The amount of serum albumin in a formulation or composition can be determined by comparison with human serum. In one aspect, the amount of serum albumin is equivalent to the amount normally present in a sample of human blood or serum.

The amount of lysozyme in the formulation or composition can be determined by comparison with the normal amount of lysozyme present in human samples. In one aspect, the amount of lysozyme is equivalent to the amount normally present in a sample of human blood or serum.

A further aspect of the invention relates to a chimeric, dimeric or fusion peptide or lysin comprising an SH3 binding domain operably linked or fused to serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or operably linked or fused to lysozyme, particularly human lysozyme. In one aspect, the chimeric, dimeric or fusion peptide or lysin comprises at least one catalytic domain and an SH3 binding domain operably linked or fused to serum albumin or lysozyme, particularly human serum albumin or human lysozyme, or an active or binding fragment or peptide thereof. In one aspect, chimeric, dimeric or fusion peptides comprising an SH3 binding domain operably linked or fused to serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin, comprising bacteria binding fragments or peptides thereof, can be used for the delivery of therapeutic agents, drugs or other payloads effective to Gram-positive bacteria, including Staphylococcus or Streptococcus bacteria. I can. In another aspect, chimeric, dimeric or fusion peptides comprising an SH3 binding domain operably linked or fused to serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin comprising a bacteria binding fragment or peptide thereof, can be used for the delivery of therapeutic agents, drugs or other payloads effective to Gram-positive bacteria, including Staphylococcus or Streptococcus bacteria. In one aspect, the therapeutic agent, drug or other payload can be lysozyme or one or more other antimicrobial peptides containing chimeric or dimeric lysins. A therapeutic agent, drug or other payload can be covalently bound, fused or operably linked to the SH3 binding domain. A therapeutic agent, drug or other payload can bind to serum albumin and can be oriented or payload by serum albumin binding or affinity.

In one aspect, the invention provides improved lysin polypeptides operably linked or fused to peptides or bacteria-binding fragments and lysin-binding fragments of serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin. In another aspect, the invention provides improved lysin polypeptides operably linked or fused to peptides or bacteria-binding and lysin-binding fragments of serum albumin, particularly human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin. In one embodiment, the dissolved element plyss2 or its effective variants include serum albumin, especially human serum arbumin, rabbits, albumin albumin, horse pepper albumin, rat serum albumin, latum (cady / cows), serum albumin, or peptide albumin. It is possible to combine the fragment or adhere to the shared bond, and the peptide or fragment can be binded to bacterial cells containing the pertragant or lensorus bacteria. In one aspect, the lysin PlySs2 or an effective variant thereof is fused or covalently attached to serum albumin, particularly human serum albumin, rabbit serum albumin, dog or horse serum albumin, or a peptide or fragment of serum albumin, which peptide or fragment is capable of binding to bacterial cells, including Staphylococcus or Streptococcus bacteria. In one aspect, the lysin polypeptide is a lysin polypeptide having an SH3-type binding domain and is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. In one aspect, the lysin polypeptide is PlySs2 (CF-301).

In one aspect, the invention provides improved lysin polypeptides operably linked or fused to peptides or bacteria-binding and lysin-binding fragments of lysozyme, particularly human lysozyme. In one aspect, the lysin polypeptide selected from PolySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, or effective killing variants thereof, is lysozyme, particularly human lysozyme, or a fragment of lysozyme capable of cleaving peptidoglycan of Gram-positive bacteria. fused to or covalently attached to. In one aspect, the lysin PlySs2 (CF-301), or an effective variant thereof capable of killing Staphylococcus and Streptococcus bacteria, is fused or covalently attached to lysozyme, particularly human lysozyme, or a fragment of lysozyme capable of cleaving the peptidoglycan of Gram-positive bacteria.

According to the present invention, there is provided a method of killing Gram-positive bacteria comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, and further contacting the bacteria with serum albumin, particularly human serum albumin, and/or lysozyme, particularly human lysozyme. In one aspect, a method of reducing a population of Gram-positive bacteria is provided comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, and further contacting the bacteria with serum albumin, particularly human serum albumin, and/or lysozyme, particularly human lysozyme. In one aspect, the bacterium is contacted with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill a Gram-positive bacterium, or a composition comprising an isolated lysin polypeptide having an SH3-type binding domain is administered to a human, and the bacterium is further contacted with serum albumin, particularly human serum albumin, and/or lysozyme, particularly human lysozyme, or serum albumin, particularly a human. Methods of treating antibiotic-resistant Staphylococcus aureus infections in humans are provided comprising further administering serum albumin and/or lysozyme, particularly human lysozyme, to the human. According to the method, serum albumin and lysin polypeptide may be administered sequentially or simultaneously, or may be administered in a composition comprising lysin polypeptide and serum albumin. According to the above methods, lysozyme and lysozyme may be administered sequentially or simultaneously, or may be administered in a composition comprising lysozyme and lysozyme. In one aspect of the methods herein, one or more serum fatty acids, particularly selected from one or more of oleate and palmitate, are further contacted or administered.

According to the present invention, it comprises contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, wherein the composition is selected from serum albumin and lysozyme, in particular one or more selected from human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin and calf (bovine/bovine) serum albumin, and human lysozyme. Methods are provided for synergistically killing Gram-positive bacteria, particularly Staphylococcus and/or Streptococcus bacteria, further comprising blood components. In one aspect, Gram-positive bacteria, particularly Staphylococcus and/or Streptococcus bacteria, comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill the Gram-positive bacteria, wherein the composition further comprises one or more blood components selected from serum albumin and lysozyme, particularly selected from human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, and human lysozyme. Methods of synergistic killing are provided. In one aspect according to the invention, there is provided a method of synergistically killing Gram-positive bacteria, particularly Staphylococcus and/or Streptococcus bacteria, comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, the composition further comprising one or more blood components selected from serum albumin and lysozyme, particularly human serum albumin and human lysozyme. In one aspect of the invention, there is provided a method of synergistically killing Staphylococcus aureus bacteria comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, wherein the composition further comprises one or more blood components selected from serum albumin and lysozyme, particularly human serum albumin and human lysozyme.

According to the present invention there is provided a method of killing Gram-positive bacteria comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain and further comprising serum albumin in an amount effective to kill Gram-positive bacteria, followed by further contacting the bacteria with lysozyme, particularly human lysozyme. In one aspect of the invention, there is provided a method of killing lysozyme-resistant or lysozyme-insensitive Staphylococcus aureus bacteria comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain and further comprising serum albumin in an effective amount to kill Gram-positive bacteria, and then further contacting the bacteria with lysozyme, particularly human lysozyme.

In one aspect of the invention, there is provided a method of killing lysozyme-resistant or lysozyme-insensitive Staphylococcus aureus bacteria comprising contacting the bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain in an amount effective to kill Gram-positive bacteria, and then further contacting the bacteria with lysozyme, particularly human lysozyme. In one aspect thereof, serum albumin, particularly human serum albumin, is additionally administered. In one aspect, the level of serum albumin and/or the binding of native serum albumin to S. aureus bacteria is first assessed or evaluated. If the serum albumin binds to the bacterium, the lysozyme is contacted, followed by administration of the lysin polypeptide, or administration in combination, eg, in a combination composition comprising lysin and lysozyme. If the serum albumin does not bind or does not bind properly to the bacterium, the serum albumin is administered first, followed by the lysin polypeptide followed by lysozyme, or alternatively the serum albumin is administered first followed by lysozyme in combination, e.g., in a combination composition comprising lysin and lysozyme. In another aspect, serum albumin is administered simultaneously, sequentially or in combination with the lysin polypeptide, followed by administration of lysozyme.

In one aspect of the method, the serum albumin can be human, rabbit, dog, horse, rat or bovine (bovine/bovine subfamily) serum albumin. In one aspect of the method, the serum albumin can be human, rabbit, dog or horse serum albumin. In one aspect of the method, the serum albumin is human serum albumin, rabbit serum albumin, dog serum albumin, horse serum albumin, rat serum albumin or bovine (bovine/bovine subfamily) serum albumin, or an active or bacteria-binding fragment or peptide thereof, particularly a Staphylococcus aureus-binding fragment or peptide of serum albumin. In one aspect of the method, the serum albumin is human serum albumin, rabbit serum albumin, dog serum albumin or horse serum albumin, or an active or bacteria-binding fragment or peptide thereof, particularly a Staphylococcus aureus-binding fragment or peptide of serum albumin. In one aspect of the method, the serum albumin is human serum albumin, or an active or bacteria-binding fragment or peptide thereof, particularly serum albumin, particularly a Staphylococcus aureus-binding fragment or peptide of human serum albumin. In one aspect of the method, the lysozyme is human lysozyme, or an active or lytic fragment or peptide thereof. In one aspect, the lysin polypeptide comprises an SH3-type binding domain. In one aspect, the lytic polypeptide is a chimeric or fusion peptide comprising an SH3-type binding domain capable of binding Gram-positive bacteria. In one aspect, the lysin polypeptide having a SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. In one aspect, the lysin polypeptide is PlySs2 (CF-301). In one aspect, the lytic polypeptide is a chimeric or fusion peptide of PlySs2 (CF-301) comprising a PlySs2 (CF-301) SH3-type binding domain capable of binding Gram-positive bacteria, particularly Staphylococcus bacteria.

In one aspect, the components, methods or assays of the invention are used with lytic polypeptides against Gram-positive bacteria, particularly lysins comprising SH3-type binding domains, including, for example, PlySs2 (CF-301) polypeptides, or variants or derivatives thereof. In one aspect, the components, methods or assays of the invention are used with lytic polypeptides, including PlySs2 (CF-301) polypeptides, or variants or derivatives thereof, against antibiotic-resistant bacteria. In one aspect, the components, methods or assays of the invention are used with lytic polypeptides, including PlySs2 (CF-301) polypeptides, or variants or derivatives thereof, against streptococcal and staphylococcal bacteria. In one aspect, the components, methods or assays of the invention are used with lytic polypeptides, including PlySs2 (CF-301) polypeptides, or variants or derivatives thereof, against antibiotic-resistant Streptococcus and/or Staphylococcus bacteria. In one aspect, the lysin polypeptide having a SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. In one aspect, the lytic polypeptide is PlySs2 (CF-301), or a derivative or variant thereof. In one aspect, the polypeptide comprises a sequence presented herein.

It has been found that lytic polypeptides, in particular lytic polypeptides with SH3 binding domains, in particular lytic polypeptides PlySs2 (CF-301), Sal, lysostaphin, are significantly more active in assays that include or are combined with the addition of serum albumin and/or lysozyme. Antimicrobial lytic polypeptides, particularly exemplary lytic polypeptides PlySs2 (CF-301), Sal, lysostaphin, are more abundant in the presence of human serum albumin (and serum albumin corresponding to other species, particularly horse, dog and rabbit serum albumin, or other species, particularly horse, dog, and rabbit serum albumin) and/or in the presence of lysozyme, particularly human lysozyme, than in broths such as cation-adjusted broth without added serum albumin. Highly active (especially up to 32- to 64- to 100-fold more active).

In one aspect of the invention, bacteriophage lysins derived from and/or effective against Streptococcus or Staphylococcus bacteria are used in methods, assays, compositions, formulations and/or constructs of the invention. In certain aspects, the lysin comprises an SH3-type bacterial binding domain. Exemplary lysin polypeptide(s) used or applicable in the present invention are provided herein, including PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, particularly PlySs2 (CF-301) lysin. be done. In one such aspect, the lysin is capable of killing strains of Staphylococcus aureus and bacteria as demonstrated herein. In one aspect, the lysin is capable of killing Staphylococcus and Streptococcus bacteria. In one aspect, the lysin is effective against antibiotic-resistant Staphylococcus aureus, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus (DRSA), and linezolid-resistant Staphylococcus aureus (LRSA). Lysins can be effective against vancomycin intermediate-sensitivity Staphylococcus aureus (VISA).

In one such aspect, the lysin is a PlySs2 (CF-301) lysin and is capable of killing strains of Staphylococcus aureus and bacteria as demonstrated herein. In one aspect, the PlySs2(CF-301) lysin is capable of killing Staphylococcus and Streptococcus bacteria. PlySs2 (CF-301) is effective against antibiotic-resistant Staphylococcus aureus such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus (DRSA) and linezolid-resistant Staphylococcus aureus (LRSA). PlySs2 (CF-301) is effective against vancomycin intermediate-resistant Staphylococcus aureus (VISA).

An isolated lysin polypeptide can include a lysin amino acid sequence presented herein or a variant thereof that has at least 80% identity, 85% identity, 90% identity, 95% identity or 99% identity to a polypeptide herein and is effective in killing Gram-positive bacteria. An isolated PlySs2 (CF-301) lysin polypeptide has at least 80% identity, 85% identity, 90% identity, 95% identity, or 99% identity to the PlySs2 (CF-301) amino acid sequence presented herein (SEQ ID NO:3), or a polypeptide herein (SEQ ID NO:3), and a variant thereof that is effective in killing Staphylococcal and Streptococcal bacteria. can include An isolated Sallysin polypeptide can include variants thereof that have at least 80% identity, 85% identity, 90% identity, 95% identity, or 99% identity to the Sal1 amino acid sequence presented herein (SEQ ID NO:5), or a polypeptide herein (SEQ ID NO:5), and that are effective in killing Staphylococcus bacteria. An isolated LysK lysin polypeptide can include variants thereof that have at least 80% identity, 85% identity, 90% identity, 95% identity, or 99% identity to the LysK amino acid sequence presented herein (SEQ ID NO:6), or a polypeptide herein (SEQ ID NO:6), and that are effective in killing Staphylococcus bacteria. An isolated lysostaphinlysin polypeptide can include a lysostaphin amino acid sequence presented herein (SEQ ID NO: 7), or a variant thereof that has at least 80% identity, 85% identity, 90% identity, 95% identity, or 99% identity to a polypeptide herein (SEQ ID NO: 7) and is effective in killing Staphylococcus bacteria or Staphylococcus and Streptococcus bacteria. An isolated lysin polypeptide, as referred to herein, is a lysin polypeptide presented herein or known in the art having at least 80% identity, 85% identity, 90% identity, 95% identity or 99% identity to a polypeptide herein or known or art-recognized, including variants thereof effective to kill Staphylococcus bacteria, or Staphylococcus and Streptococcus bacteria. It may contain an amino acid sequence.

In any such method(s) above, the bacterium is Staphylococcus aureus, Listeria monocytogenes, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi , Streptococcus equi zoo, Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS), Streptococcus sanguinis, Streptococcus gordonii, Streptococcus Cassus dysgalactiae (Streptococcus dysgalactiae), G
It can be selected from group streptococci, group E streptococci, Enterococcus faecalis and Streptococcus pneumonia.

According to any of the methods of the invention, the bacterium can be an antibiotic resistant bacterium. The bacterium can be methicillin-resistant Staphylococcus aureus (MRSA), vancomycin intermediate-resistant Staphylococcus aureus (VISA), vancomycin-resistant Staphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus (DRSA), or linezolid-resistant Staphylococcus aureus (LRSA). Susceptible bacteria can be clinically relevant or pathogenic bacteria, especially for humans. In one aspect of the method(s), the lysin polypeptide(s) is effective to kill Staphylococcus, Streptococcus, Enterococcus and Listeria bacterial strains.

In additional aspects or embodiments of the methods and compositions provided herein, another different staphylococcal-specific lysin is used herein alone or in combination with the lysins provided herein, including the PlySs2 (CF-301) lysins provided and described herein. In one such aspect or embodiment of the methods and compositions provided herein, one or more lysins selected from Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin are used alone or in combination with the PlySs2 (CF-301) lysin presented and described herein. . In one aspect or embodiment of the methods and compositions provided herein, one or more lysins selected from Sal lysin, LysK lysin, Lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin are combined together as provided and described herein. In one aspect or embodiment of the methods and compositions provided herein, one or more SH3-binding domains of a lysin selected from Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin are used in combination with at least one other catalytic domain of another lysin, including those presented and described herein.

Other objects and advantages will become apparent to those skilled in the art from a consideration of the following description, which proceeds with reference to the accompanying illustrative drawings.

FIG. 2 shows enhanced lytic activity of PlySs2 (CF-301) in human blood matrix. Composite time-kill curves of PlySs2 (CF-301) against S. aureus strain MW2 tested in (A) human serum and 4 pooled samples from 16 individuals, (B) human whole blood from 10 individuals, (C) MHB (10 replicates), and (D) pooled BALB/c mouse serum are compared to buffer controls. Mean values (±SEM) are shown for each time point. FIG. 1 shows a study of the effects of human blood on various named Staphylococcus aureus strains. Composite time-kill curves of PlySs2 (CF-301) against indicated S. aureus strains tested using 5 replicates in either MHB or pooled human serum are compared to buffer controls. Mean values (±SEM) are shown for each time point. FIG. 4 shows the effect of human blood on another lysin-like protein (lysostaphin) and a small molecule antibiotic (vancomycin). Composite time-kill curves for indicated compounds against S. aureus strain MW2 are compared to buffer controls. A and B, Lysostaphin tested with 5 replicate samples in either MHB or pooled human serum, respectively. C and D, Vancomycin tested with 5 replicate samples in either MHB or pooled human serum, respectively. Mean values (±SEM) are shown for each time point. FIG. 2 shows enhanced lytic activity of PlySs2 (CF-301) in animal blood matrices. PlySs2 (CF-301) against S. aureus strain MW2 tested in (A) fetal bovine serum (3 replicates each for 3 different lots), (B) Sprague Dawley serum blood (5 replicates each for 2 pooled lots), (C) rabbit serum (5 replicates each for 2 pooled mixed-breed lots) and (D) beagle dog serum from 8 individual animals. Compare the composite time-kill curve of . Mean values (±SEM) are shown for each time point. MIC distribution of PlySs2 (CF-301) for S. aureus MW2 in different human blood matrices. Histograms are shown for analyzes performed on multiple different samples including individual and pooled whole blood (n=13), serum (n=32) and plasma (n=15). Each sample is described in Supplementary Table 1. The MIC is plotted on the x-axis and the number of patient samples with a particular MIC is plotted on the y-axis. Figure 2 demonstrates that PlySs2 (CF-301) exhibits potent synergy with other antimicrobial agents in human serum. Composite time-kill curves for the indicated single agents (concentrations in brackets) against S. aureus strain MW2 are compared to both the buffer control and a combination of both agents (each indicated single agent concentration). Mean values (±SEM) based on triplicate assays are shown for each time point. AC, PlySs2 (CF-301) tested at sub-MIC amounts in the presence of a constant amount of DAP. D, PlySs2 (CF-301) and lysostaphin tested using amounts below the MIC. FIG. 2 shows colorimetric MIC determination of S. aureus MW2 using Alamar Blue™ at 18 hours. Addition of Alamar blue dye (Resazurin) to each well for 2 hours allows assessment of viability (pink) and cell death (blue) over a log10 dilution range. A, Viability assays in MHB, HuS and MuS. Samples were assayed in HuS either untreated or pretreated with proteinase K-agarose beads for 3 hours. As a control for protease carryover, proteinase K pretreated serum was diluted 3:4 in untreated serum prior to analysis. B, Viability assay in HuS pretreated for 30 min at various temperatures. C, Checkerboard analysis of PlySs2 (CF-301) in combination with HuS using MHB as diluent. Red squares indicate HuS dilutions at which the enhancer effect is attenuated. FIG. 4 shows the effect of HuLYZ and HSA on PlySs2 (CF-301) activity. A presents a time-kill assay combining sub-MIC amounts of lysin with various huLYZ concentrations. B and C show a second in vitro assay based on the loss of optical density in treated cultures with the addition of HuLYZ (B) and HSA (C). FIG. 2 shows Western blot studies using anti-PlySs2 (CF-301) antibody. Labeling of PlySs2 (CF-301) in the presence and absence of rHSA (red). Data (not shown) do not label either PIyG ^GFP or PIyC ^AF at 25 μg/ml with or without HSA. Effect of different serotypes (and MHB) pre-incubation on subsequent PlySs2 (CF-301) labeling (red). Labeling with HuLYZ (green) in the presence and absence of PlySs2(CF-301) (1-0.25x MIC). TEM analysis of S. aureus strain MW2 treated with PlySs2 (CF-301) for 15 minutes in either human serum (HuS) or MHB. FIG. 4 shows the efficacy of various PlySs2 (CF-301) dosing regimens in addition to daptomycin in rat (A) and rabbit (B) infective endocarditis (IE) models. Data are plotted as treatment plan versus mean log ₁₀ CFU/g of tissue for each dose group. Median ± SEM are also shown. FIG. 2 shows AUC values and AUC dose proportionality for various PlySs2 (CF-301) dosing regimens in rats and rabbits.

According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques within the skill of those skilled in the art can be used. Such techniques are explained fully in the literature. See, for example, Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989), "Current Protocols in
Molecular Biology" Volumes I-III [Ausubel, RM, ed. (1994)], "Cell Biology: A
Laboratory Handbook" Volumes I-III [JE Celis, ed. (1994))], "Current Protocols in Immunology" Volumes I-III [Coligan, JE, ed. (1994)], "Oligonucleotide Synthesis" (MJ Gait ed. 1984), "Nucleic Acid Hybridization" [BD Hames & SJ Higgins eds. (1985)], "Transcription And Translation "[BD Hames & SJ Higgins, eds. (1984)], "Animal Cell Culture" [RI Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)], B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

Additionally, in accordance with the present invention, various terms may be used and referred to and have definitions or explanations presented herein and below.

In a general aspect of the invention, it has been found that certain lysins, particularly lysins with SH-3 type binding domains, including PlySs2 (CF-301) lysins, are more effective in human blood, serum and plasma than in artificial media. Increases and enhancements of activity up to 100-fold were identified. In addition to human sera, potentiation is also observed in rabbit, dog and horse sera. An intermediate effect is observed in rat serum. Mouse sera show no enhancing effect. We hypothesize, and now demonstrate, that certain phage lysins, particularly lysins with SH-type 3 binding domains, including PlySs2 (CF-301), are capable of favorable antimicrobial interactions with one or more components in blood or blood fractions.

According to the present invention, blood components have been identified that exhibit synergy with lysins, including PlySs2 (CF-301). In one aspect, serum albumin, particularly human serum albumin, enhances the antibacterial activity of lysins, including PlySs2 (CF-301) lysins. Serum albumin is the most abundant protein in human plasma, accounting for about half of the serum proteins. The reference range for albumin concentration in serum is approximately 35 g/L to 50 g/L or 3.5 g/dL to 5.0 g/dL. Albumin has a serum half-life of approximately 20 days.

この配列中の６０９個のアミノ酸のうち、５８５個のアミノ酸が血液中に存在する最終生成物において観察され、シグナルペプチド（１～１８）及びプロペプチド部分を含む最初の２４個のアミノ酸（ここではイタリック体とし、下線を引いた）は、翻訳後に切断される。 The gene for albumin is located on chromosome 4 and is divided into 15 exons that are symmetrically arranged within three domains that are thought to arise from triplication of a single primitive domain. Among other functions, albumin transports hormones, fatty acids and other compounds, buffers pH and maintains oncotic pressure. The amino acid sequence of human serum albumin (Uniprot P02768) is as follows (SEQ ID NO: 1):

Of the 609 amino acids in this sequence, 585 amino acids are observed in the final product present in blood, and the first 24 amino acids (here italicized and underlined), including the signal peptide (1-18) and propeptide portions, are post-translationally cleaved.

In a further aspect, lysozyme, particularly human lysozyme, enhances the antibacterial activity of lysins, including PlySs2 (CF-301) lysins. Lysozyme, also known as muramidase or N-acetylmuramidoglycan hydrolase, is an antimicrobial enzyme produced by animals that is part of the innate immune system. Lysozyme catalyzes the hydrolysis of 1,4-β-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan, a major component of Gram-positive bacterial cell walls, resulting in compromised bacterial cell wall integrity and bacterial lysis. In particular, Stapylococcus aureus bacteria are completely lysozyme-resistant, contributing significantly to their persistence and successful colonization of skin and mucosal areas of humans and animals (Bera, A et al (2004) Molecular Microbiology 55(3):778-787, Pushkaran, AC et al (2015) J Chem Inf Model 55(4):760-770).

Human lysozyme is a 148 amino acid protein containing an 18 amino acid signal peptide sequence, resulting in a 129 amino acid mature protein. The sequence of human lysozyme corresponds to Uniprot P61626 and Genbank NP_000230 and is as follows (SEQ ID NO: 2) (signal peptide italicized and underlined):

According to the present invention, the relevant lysin polypeptides used in the present invention may in particular be lysin polypeptides with SH3-type binding domains. The Src homology 3 (SH3) enzymatic domain has a characteristic β-barrel fold consisting of five or six β-strands arranged as two tightly packed anti-parallel β-sheets. Classical SH3 domains are commonly found in proteins that interact with other proteins and mediate the assembly of specific protein complexes, including by binding to proline-rich peptides in their respective binding partners. Many SH3-binding epitopes of proteins have proline-containing sequence motifs. SH3 domains and sequences are described and reviewed in the prior art, including Whisstock, J.C. and Lesk, A.M. (1999) TIBS 24:132-133 and Ponting, C.P. et al (1999) J Mol Biol 289:729-745.

In one aspect thereof, the lysin polypeptide having an SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. In one aspect, the lysin polypeptide is PlySs2.

The terms "PlySs lysin(s)", "PlySs2 lysin", "PlySs2", "PlySs2(CF-301)", "PlySs2/CF-301", "CF-301", "CF301" and any variations not specifically mentioned may be used interchangeably herein and are used throughout this application and claims. When used, it refers to a proteinaceous substance comprising a protein or proteins, and extends to proteins having the amino acid sequence data described herein and presented below, as well as proteins having the profile of activity described herein and in the claims. Thus, proteins that exhibit substantially similar or altered activity are also contemplated. These modifications may be intentional, such as those obtained by site-directed mutagenesis, or may be accidental, such as those obtained by mutation in the host that is the producer of the complex or its specified subunits. The terms "PlySs lysin(s)", "PlySs2 lysin", "PlySs2", "PlySs2(CF-301)", "PlySs2/CF-301", "CF-301", "CF301" also refer to the proteins specifically recited herein, as well as all substantially homologous analogs, fragments or truncations. s), and is intended to include allelic variations within its scope. PlySs2 lysins are described in US Pat. Gilmer et al also described a PlySs2 lysin (Non-Patent Document 4). The PlySs2 (CF-301) amino acid sequence is provided below (SEQ ID NO:3). The N-terminal CHAP domain (cysteine-histidine amidohydrolase/peptidase) of the PlySs2 (CF-301) polypeptide lysin (starting with LNNV... ending with HYIT) and the C-terminal SH3-type binding domain (starting with RSYR... ending with YVAT) are underlined below.

The terms "ClyS", "ClyS lysin" refer to the chimeric lysin ClyS that has activity against Staphylococcus aureus, including Staphylococcus aureus, and is described in detail in US Pat. ClyS does not have an SH3-type binding domain. An exemplary amino acid sequence for ClyS is provided below (SEQ ID NO:4).

Sal lysin, alternatively designated Sal1, is described in several Yoon et al references, including US Pat. Nos. 8,232,370, 8,377,431 and 8,377,866. An exemplary Sal1 sequence is provided below (SEQ ID NO:5).

LysK lysin is an antistaphylococcal lysin and contains an amidase, a CHAP domain and an SH3-type binding domain. LysK is described in O'Flaherty S et al (2005) J Bacteriol 187(20):7161-7164. Fusion proteins, particularly fusions of LysK and lysostaphin, are described by Donovan et al in US Pat. No. 8,568,714. Ply187
Chimeric/chimeric lysins with an endopeptidase domain and a LysK SH3 cell wall binding domain are described in US Patent Application No. 13/432,758 and WO2013/149010. The LysK sequence is presented below (SEQ ID NO:6).

It is worth noting that the sequences of Sal-1 and LysK are very similar, differing only at amino acids 26, 114 and 485, which are underlined in each sequence above.

Lysostaphin and recombinant lysostaphin are described in various references including Sloan GL et al (1982) Int J Sys Bacteriol 32:170-174 and Oldham ER and Daley MJ (1991) J Dairy Science 74:1127-1131. A cloned lysostaphin sequence from Staphylococcus simulans is described in Recsei PA et al (1987) PNAS USA 84:1127-1131 and presented in US Pat. No. 4,931,390. Mature lysostaphin consists of 246 amino acid residues. The preprotein contains three distinct regions, a signal peptide (about 30 aa to 38 aa) typical of precursor proteins, a hydrophilic and highly ordered protein domain (296 aa) containing 14 repeats and a hydrophobic mature lysostaphin. An exemplary sequence for lysostaphin is provided below (SEQ ID NO:7). The mature sequence is in bold.

ALE-1 lysin is a lysostaphin homolog, Liu JZ et al (2006) J Biol
Chem 281:549-558.

Phil bacteriophage lysins contain two hydrolase domains, endopeptidase and amidase, and an SH3B-type binding domain, and are known and described in the art, including Donovan DM et al (2006) FEMS Microbiol Lett 265(1):133-139. LysH5 lysin is also known, composed of three domains, two hydrolase domains, endopeptidase and amidase, and an SH3B-type binding domain, described for example in Obeso JM et al (2008) Int J Food Microbiol 128(2):212-228. Phage lysin MV-L is described in Rashel M et al (2007) J Infect Dis 196(8):1237-1247. MV-L lysins are composed of two catalytic domains (endopeptidase and amidase domains) linked to a single cell wall targeting (CWT) domain, a type of binding domain. Unless otherwise indicated, references herein to "binding domain" include the CWT domain. The MV-L CWT domain shows homology to the SH3b-like domain as well as the staphylolytic enzyme lysostaphin.
The SH3b-like domain binds to peptide bridges (pentalysin) in the staphylococcal cell wall.

Polypeptides and Lytic Enzymes "Lytic enzyme" includes any bacterial cell wall lytic enzyme that, under suitable conditions, kills one or more bacteria over a relevant period of time. Examples of lytic enzymes include, but are not limited to, various amidase cell wall lytic enzymes.

A "sugar lytic enzyme" includes a lytic enzyme capable of killing at least one or more Streptococcus suis bacteria under suitable conditions and over a relevant period of time.

"Bacteriophage lytic enzyme" refers to a lytic enzyme extracted or isolated from a bacteriophage or a synthetic lytic enzyme having a similar protein structure that maintains lytic enzyme functionality.

Lytic enzymes are capable of specifically cleaving the bonds present in the peptidoglycan of bacterial cells and destroying the bacterial cell wall. It is also currently hypothesized that bacterial cell wall peptidoglycan is highly conserved in most bacteria and that cleavage of only a few bonds can disrupt the bacterial cell wall. The bacteriophage lytic enzyme can be an amidase, but other types of enzymes are possible. Examples of lytic enzymes that cleave these bonds are various amidases such as muramidase, glucosaminidase, endopeptidase or N-acetyl-muramoyl-L-alanine amidase. Fischetti et al (1974) reported that the C1 streptococcal phage lysin enzyme is an amidase. Garcia et al (1987, 1990) reported that the Cpl lysin from Streptococcus pneumoniae derived from the Cp-1 phage is lysozyme. Caldentey and Bamford (1992) report that the lytic enzyme from the phi 6 Pseudomonas phage is an endopeptidase that separates peptide bridges formed by meso-diaminopimelic acid and D-alanine. E. coli T1 and T6
Phage lytic enzymes are amidases, like the lytic enzyme (ply) from Listeria phage (Loessner et al, 1996). Other lytic enzymes capable of cleaving bacterial cell walls are also known in the art.

A "lytic enzyme genetically encoded by a bacteriophage" includes a polypeptide capable of killing a host bacterium, eg, by having at least some cell wall lytic activity against the host bacterium. The polypeptide can have a sequence that encompasses the native sequence lytic enzyme and variants thereof. The polypeptides can be isolated from a variety of sources, such as bacteriophage (“phage”), or can be prepared by recombinant or synthetic methods, such as those described by Garcia et al and presented herein. The polypeptide may contain a choline binding moiety at the carboxyl terminus and may be characterized by an enzymatic activity capable of cleaving cell wall peptidoglycan at the amino terminus (such as an amidase activity acting on an amide bond in peptidoglycan). A lytic enzyme containing multiple enzymatic activities, such as the PlyGBS lysin, for example two enzymatic domains, has been described. Generally, lytic enzymes have a molecular weight between 25,000 and 35,000 Daltons and may contain a single polypeptide chain, although this may vary depending on the enzyme chain. Molecular weight can be most conveniently determined by assay on denaturing sodium dodecyl sulfate gel electrophoresis and comparison with molecular weight markers.

A "native sequence phage-associated lytic enzyme" includes a polypeptide having the same amino acid sequence as an enzyme derived from bacteria. Such native sequence enzymes can be isolated or produced by recombinant or synthetic means.

The term "native sequence enzyme" encompasses naturally occurring forms (eg, alternatively spliced or modified forms) and naturally occurring variants of the enzyme. In one embodiment of the invention, the native sequence enzyme is a mature or full-length polypeptide genetically encoded by a gene derived from a bacteriophage that is specific for or capable of killing streptococci. Of course Lopez et al., Microbial Drug Resistance 3: 199-211 (1997); Garcia et al., Gene 86: 81-88 (1990); Garcia et al., Proc. Natl. Acad. : 914-918 (1988), Garcia et al., Streptococcal Genetics (JJ Ferretti and Curtis eds., 1987), Lopez et al., FEMS Microbiol. Lett. 100: 439-448 (1992), Romero et al., J. Bacteriol. Many variants are possible and known, as recognized in the literature such as Ronda et al., Eur. J. Biochem. 164: 621-624 (1987) and Sanchez et al., Gene 61: 13-19 (1987). The content of each of these references, particularly the sequence listing and accompanying text comparing sequences, including statements regarding sequence homology, is specifically incorporated herein by reference in its entirety.

A "variant sequence lytic enzyme" includes a lytic enzyme characterized by a polypeptide sequence that differs from a naturally occurring lytic enzyme, but retains functional activity. A lytic enzyme may, in some embodiments, be genetically encoded by a bacteriophage specific to a bacterium, such as Streptococcus, which has specific amino acid sequence identity with the lytic enzyme sequence(s) herein presented or referred to herein. For example, in some embodiments, functionally active lytic enzymes can kill streptococci and other susceptible bacteria presented herein, for example, by disrupting the bacterial cell wall. An active lytic enzyme may have 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% amino acid sequence identity with the lytic enzyme sequence(s) herein presented or referred to herein. Such phage-associated lytic enzyme variants include, for example, lytic enzyme polypeptides in which one or more amino acid residues have been added or deleted at the N-terminus or C-terminus of the lytic enzyme sequence(s) herein presented or referred to herein. In certain aspects, the phage-associated lytic enzyme has at least about 80% or 85% amino acid sequence identity, particularly at least about 90% (eg, 90%) amino acid sequence identity with a native phage-associated lytic enzyme sequence. Most specifically, the phage-associated lytic enzyme variant has at least about 95% (e.g., 95%) amino acid sequence identity with the native phage-associated lytic enzyme sequence(s) herein presented or referred to herein.

"Percent amino acid sequence identity" for a specified phage-associated lytic enzyme sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the phage-associated lytic enzyme sequence after aligning the sequences in the same reading frame and introducing gaps as necessary to achieve maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.

"Percent nucleic acid sequence identity" with respect to the phage-associated lytic enzyme sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in the phage-associated lytic enzyme sequence after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity.

To determine the percent identity of two nucleotide or amino acid sequences, the sequences are aligned for optimal comparison (eg, gaps may be introduced into the sequence of the first nucleotide sequence). The nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared between the sequences (ie, % identity = number of identical positions/total number of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm used to compare two sequences is that of Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such algorithms are incorporated into the NBLAST program which can be used to identify sequences having the desired identity to the nucleotide sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res, 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, NBLAST) can be used. See programs offered by the National Center for Biotechnology Information, the National Library of Medicine, and the National Institutes of Health. In one embodiment, the parameters for sequence comparison can be set at W=12. Parameters can also be changed (eg W=5 or W=20). The value "W" determines the number of contiguous nucleotides that must be identical for the program to identify two sequences as having identical regions.

A "polypeptide" includes a polymer molecule composed of linearly joined amino acids. A polypeptide, in some embodiments, can correspond to a molecule encoded by a naturally occurring polynucleotide sequence. Polypeptides may contain conservative substitutions in which naturally occurring amino acids are replaced with amino acids having similar properties, but such conservative substitutions do not alter the function of the polypeptide (see, e.g., Lewin "Genes V" Oxford University Press Chapter 1, pp. 9-13 1994).

The term "modified lytic enzyme" includes shuffled and/or chimeric lytic enzymes.

Phage lytic enzymes specific for bacteria infected with particular phages have been found to effectively and efficiently degrade the cell walls of the bacteria in question. Lytic enzymes lack protease activity and are therefore believed to be non-destructive to mammalian proteins and tissues when present during digestion of bacterial cell walls. "Rapid Killing of Streptococcus pneumoniae with a Bacteriophage Cell Wall Hydrolase," Science, 294: 2170-2172 (Dec. 7,
2001) (Non-Patent Document 3) and supplementary material published online in the journal Science (incorporated herein by reference in its entirety), purified pneumococcal bacteriophage lytic enzymes such as Pal are capable of killing a variety of pneumococcal species. Loeffler et al. showed in these experiments that the lytic enzyme Pal was able to kill 15 clinical strains of Streptococcus pneumoniae in vitro within seconds of contact, including the most commonly isolated serogroups and penicillin-resistant strains. Treatment of mice with Pal was also able to dose-dependently eliminate or significantly reduce serotype 14 nasal carriage. Furthermore, the action of Pal was found to be similar to other phage lytic enzymes but somewhat specific to the target pathogen, unlike antibiotics, which may leave the normal flora essentially intact (MJ Loessner, G. Wendlinger, S. Scherer, Mol.
Microbiol 16, 1231-41. (1995), incorporated herein by reference). In contrast, certain lysin polypeptides of the present invention have remarkably broad and clinically significant bacterial killing profiles. For example, the isolated Streptococcus suis lysin PlySs2 is effective in killing Streptococcus suis, as well as various other streptococcal strains including Group B Streptococcus (GBS), Staphylococcus strains including Staphylococcus aureus, Enterococcus spp. and Listeria spp. The lysins of the present invention can exhibit broad bacterial cell killing across Staphylococcus and/or Streptococcus strains or bacteria.

The lytic enzyme(s) or polypeptide(s) may be truncated, chimeric, shuffled or "native" and may be combinations. The related US Pat. No. 6,200,000 is hereby incorporated by reference in its entirety. "Modified" lytic enzymes can be made in a number of ways. In one embodiment, the modified lytic enzyme gene is placed in a transferable or mobilizable vector, preferably a plasmid, and the plasmid is cloned into an expression vector or expression system. Expression vectors for producing the lysin polypeptides or enzymes of the invention may be suitable for E. coli, Bacillus or many other suitable bacteria. The vector system may be a cell-free expression system. All of these methods of expressing a gene or genes are known in the art.

A "chimeric protein" or "fusion protein" comprises all or a (preferably biologically active) portion of a polypeptide of the invention operably linked to a heterologous polypeptide. A relevant biologically active portion may be a catalytic domain. A relevant biologically active portion may be a binding domain. Chimeric proteins or peptides are made, for example, by combining two or more proteins with two or more active sites. Chimeric proteins and peptides can act independently on the same or different molecules and thus have the potential to treat two or more different bacterial infections simultaneously. Thus, chimeric proteins may combine a single binding domain, such as an SH3-type binding domain, with two or more catalytic domains. Chimeric proteins and peptides can also be used to treat bacterial infections, e.g., by cleaving the cell wall at two or more locations through two (or more) catalytic domains or two (or more) catalytic activities, potentially resulting in more rapid or effective (or synergistic) killing from a single lysin molecule or chimeric peptide.

A "heterologous" region of a DNA or peptide construct is a distinguishable segment of DNA within a larger DNA molecule or peptide within a larger peptide molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene is usually flanked by DNA that does not flank the mammalian genomic DNA in the original organism's genome. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (eg, a cDNA in which the genomic coding sequence contains introns or synthetic sequences having different codons than the native gene). Allelic variation or spontaneous mutational events do not give rise to heterologous regions of DNA or peptides as defined herein.

The term "operably linked" means that a polypeptide of the present disclosure and a heterologous polypeptide are fused in-frame. Heterologous polypeptides can be fused to the N-terminus or C-terminus of a polypeptide of the disclosure. Chimeric proteins are produced enzymatically by chemical synthesis or by recombinant DNA technology. A number of chimeric lytic enzymes have been generated and studied. Chimeric lytic genes EL constructed from bacteriophage phi X174 and MS2 lytic proteins E and L, respectively, were subjected to internal deletions to generate a series of new EL clones with altered lytic or killing properties. In this study, we investigated the lytic activity of the endotruncated versions of the parental genes E, L, EL and EL and characterized different lytic mechanisms based on structural differences in the different transmembrane domains. Electron microscopy of the cytoplasmic and pericellular spaces and the release of marker enzymes revealed that two different lytic mechanisms could be distinguished depending on the permeabilization of proteins in either the inner or inner and outer membranes of E. coli (FEMS Microbiol. Lett. (1998) 164(1):159-67 (incorporated herein by reference)). One example of a useful fusion protein is a GST fusion protein in which a polypeptide of the disclosure is fused to the C-terminus of GST sequences. Such chimeric proteins can facilitate the purification of recombinant polypeptides of this disclosure.

In another embodiment, the chimeric protein or peptide contains a heterologous signal sequence at its N-terminus. For example, the native signal sequence of a polypeptide of the disclosure can be removed and replaced with a signal sequence derived from another protein. For example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992, incorporated herein by reference). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, Calif.). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretion signal (Sambrook et al., supra) and the protein A secretion signal (Pharmacia Biotech; Piscataway, NJ).

Fusion proteins can combine a lysin polypeptide with a protein or polypeptide that has a different ability or that confers additional abilities or additional characteristics to the lysin polypeptide. Fusion proteins may be immunoglobulin fusion proteins in which all or part of a polypeptide of the present disclosure is fused to sequences derived from a member of the immunoglobulin protein family. Immunoglobulins can be antibodies, eg, antibodies directed against surface proteins or epitopes of susceptible or target bacteria. Immunoglobulin fusion proteins can be incorporated into pharmaceutical compositions and administered to a subject to inhibit the interaction between ligands (soluble or membrane-bound) and proteins on the surface of cells (receptors), thereby inhibiting signal transduction in vivo. Immunoglobulin fusion proteins can alter the bioavailability of the cognate ligands of the polypeptides of this disclosure. Inhibition of ligand/receptor interactions may be therapeutically useful for the treatment of both bacteria-associated diseases and disorders that modulate (ie promote or inhibit) cell survival. In addition, the immunoglobulin fusion proteins of this disclosure can be used as immunogens to raise antibodies to the polypeptides of this disclosure in a subject, to purify ligands, and in screening assays to identify molecules that inhibit the interaction of receptors and ligands. Chimeric and fusion proteins and peptides of the disclosure can be made by standard recombinant DNA methods.

The fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, anchor primers can be used to PCR amplify the gene fragment, resulting in complementary overhangs between two consecutive gene fragments, which can then be annealed and re-amplified to generate chimeric gene sequences (i.e., see Ausubel et al., supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (ie, a GST polypeptide). A nucleic acid encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the invention.

As used herein, shuffled proteins or peptides, gene products, or peptides of two or more related phage proteins or protein peptide fragments were randomly cleaved to reconstruct more active or specific proteins. Shuffled oligonucleotide, peptide or peptide fragment molecules are selected or screened to identify molecules with desired functional properties. This method is described, for example, in Stemmer, US Pat. No. 6,132,970 (method for shuffling polynucleotides), Kauffman, US Pat. No. 5,976,862 (evolution by codon-based synthesis) and Huse, US Pat. No. 5,808,022 (direct codon synthesis). The contents of these patents are incorporated herein by reference. Shuffling can be used to create proteins that are, for example, up to 10- to 100-fold more active than the template protein. Template proteins are selected from a variety of different lysin proteins. Shuffle proteins or peptides constitute, for example, one or more binding domains and one or more catalytic domains. Each binding or catalytic domain is derived from the same or different phage or phage protein. A shuffle domain is either an oligonucleotide-based molecule as a gene or gene product translatable into peptide fragments, either alone or in combination with other genes or gene products, or is a peptide-based molecule. Gene fragments include any molecule of DNA, RNA, DNA-RNA hybrids, antisense RNA, ribozymes, ESTs, SNIPs, and other oligonucleotide-based molecules that alone or in combination with other molecules yield an oligonucleotide molecule capable or incapable of being translated into a peptide.

Improved or modified forms of the proteins or peptides and peptide fragments disclosed herein include proteins or peptides and peptide fragments either chemically synthesized or made by recombinant DNA methods, or both. These techniques include, for example, chimerization and shuffling. If the protein or peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, ie separated from chemical precursors or other chemicals involved in the synthesis of the protein. Accordingly, such preparations of protein contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

A polypeptide signal sequence can facilitate transmembrane movement of the proteins and peptides and peptide fragments of the present disclosure to and from the mucosa by facilitating secretion and isolation of the secretory or other protein of interest. Signal sequences are generally characterized by a core of hydrophobic amino acids that are cleaved from the mature protein upon secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature protein as it passes through the secretory pathway. Thus, the present disclosure can relate to the described polypeptides with signal sequences, as well as the signal sequences themselves, and polypeptides in the absence of signal sequences (ie, cleavage products). A nucleic acid sequence encoding a signal sequence of the disclosure can be operably linked in an expression vector to a protein of interest, such as a protein that is not normally secreted or otherwise difficult to isolate. The signal sequence directs secretion of the protein from, for example, a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, a signal sequence can be linked to the protein of interest using a sequence that facilitates purification, such as a sequence with a GST domain.

The invention also relates to other variants of the polypeptides of the invention. Such variants may have altered amino acid sequences that can function as either agonists (mimetics) or as antagonists. Variants may be generated by mutagenesis, ie discrete point mutations or truncations. Agonists may retain substantially the same or part of the biological activity of the naturally occurring form of the protein. A protein antagonist can inhibit one or more activities of a naturally occurring form of a protein, for example, by competitively binding to downstream or upstream members of a cell signaling cascade involving the protein of interest. Thus, specific biological effects can be induced by treatment with limited-function variants. Treatment of a subject with a variant that possesses some of the biological activity of the naturally occurring form of the protein may have fewer side effects in the subject than treatment with the naturally occurring form of the protein. Variants of the proteins of the disclosure that function as either agonists (mimetics) or antagonists can be identified by screening combinatorial libraries of mutants, i.e. truncation mutants, of the proteins of the disclosure for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a mixed-strand gene library. Mixed-strand libraries of variants can be generated, for example, by enzymatically ligating mixtures of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences can be expressed as individual polypeptides or, alternatively, as a larger set of fusion proteins (i.e., for phage display). There are a variety of methods that can be used to generate a library of potential variants of the polypeptides of this disclosure from degenerate oligonucleotide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (i.e., see Narang (1983) Tetrahedron 39:3, Itakura et al. (1984) Annu. Rev. Biochem. 53:323, Itakura et al. (1984) Science 198:1056, Ike et al. (1983) Nucleic Acid Res. 11:477, all cited). made part hereby).

In addition, libraries of fragments of coding sequences of polypeptides of the present disclosure can be used to generate mixed chain populations of polypeptides for screening and subsequent selection of mutants, active fragments or truncations. For example, the library of the code array fragment is that nicking occurs only about once per molecule per molecule, which is a cord sequence of the cord sequence that is interested in a two -strag arrangement of the cord sequence that is interested in it with a nucleuses, degenerate the double -stranded DNA, regenerates DNA, and derives the sense of the sense / antisense derived from different kicking products. It can be generated by forming a two -stranded DNA that can be obtained, removing a single -strag from a double chain that is re -formed by the processing of S1 nucleese, and rigating the obtained fragment library into an expression vector. By this method, expression libraries can be obtained that encode N-terminal and internal fragments of the protein of interest of various sizes. Several techniques are known in the art for screening gene products of combinatorial libraries generated by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. The most widely used techniques amenable to high-throughput analysis for screening large gene libraries typically involve cloning the gene library into a replicable expression vector, transforming the resulting library of vectors into appropriate cells, and expressing the combinatorial genes under conditions where detection of the desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional mutants in a library, can be used in conjunction with screening assays to identify variants of the proteins of the disclosure (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327- 331). Immunologically active portions of proteins or peptide fragments include regions that bind antibodies that recognize phage enzymes. In this context, the minimal portion of a protein (or nucleic acid encoding a protein) according to embodiments is an epitope recognizable as being specific to the phage that make up the lysin protein. Thus, a minimal polypeptide (and associated nucleic acid encoding a polypeptide) that can be expected to bind a target or receptor, such as an antibody, and useful in some embodiments, can be 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, or 100 amino acids in length. Small sequences as short as 8, 9, 10, 11, 12 or 15 amino acids in length certainly contain sufficient structure to act as targets or epitopes, but shorter sequences as short as 5, 6 or 7 amino acids in length may under some circumstances represent target or epitope structure and be of value in one embodiment. Thus, the minimal portion of the protein(s) or lysin polypeptides presented herein, including those presented in SEQ ID NO:3 or SEQ ID NOS:5-6, includes polypeptides as small as 5, 6, 7, 8, 9, 10, 12, 14 or 16 amino acids in length.

A biologically active portion of a protein or peptide fragment of an embodiment includes a polypeptide comprising an amino acid sequence sufficiently identical to or derived from an amino acid sequence of a phage protein of the present disclosure that comprises fewer amino acids than the full-length protein of the phage protein and that exhibits at least one activity of the corresponding full-length protein, as described herein. Biologically active portions typically comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein or protein fragment of the disclosure can be a polypeptide that is, for example, 10, 25, 50, 100 fewer or more amino acids in length. In addition, other biologically active portions in which other regions of the protein have been deleted or added can be produced by recombinant methods and evaluated for one or more of the functional activities of the native form of the polypeptides of embodiments.

As will be appreciated by those skilled in the art, homologous proteins and nucleic acids can be made that share functionality with such small proteins and/or nucleic acids (or protein and/or nucleic acid regions of larger molecules). Short regions of such small molecules and larger molecules that may be specifically homologous are contemplated as embodiments. Preferably, such regions of interest have at least 50%, 65%, 75%, 80%, 85%, preferably at least 90%, 95%, 97%, 98% or at least 99% homology relative to the lysin polypeptides presented herein, including, for example, SEQ ID NO:3 and SEQ ID NOs:5-6, as shown or referred to. These percent homology values do not include changes due to conservative amino acid substitutions.

Two amino acid sequences are "substantially homologous" if at least about 70% (preferably at least about 80%, at least about 85%, preferably at least about 90% or 95%) of the amino acid residues are identical or exhibit conservative substitutions. The sequences of equivalent lysins, such as equivalent PlySs2 lysins or equivalent Sal or LysK lysins, are substantially homologous if one or more, or some, or up to 10%, or up to 15%, or up to 20% of the amino acids of the lysin polypeptide are replaced with similar or conservative amino acid substitutions, equivalent lysins may have the activity, antibacterial, antimicrobial activity of the lysins, such as PlySs2 and/or Sal or LysK lysins disclosed herein. It has a profile of efficacy and/or bacterial specificity.

The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue as long as the desired functional property of immunoglobulin binding is retained by the polypeptide. _NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with the standard polypeptide nomenclature of J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are provided in the table of correspondence below.

Correspondence table symbol Amino acid 1-letter 3-letter Y Tyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A Ala Alanine S Ser Serine II Ile Isoleucine L Leu Leucine T Thr Threonine V Val Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine E G lu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NA Asn asparagine C Cys cysteine

Note that all amino acid residue sequences are represented herein by formulas whose left-to-right orientation is the conventional direction from amino-terminus to carboxy-terminus. Additionally, note that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to the additional sequence of one or more amino acid residues. The above table is presented to correlate the three-letter and one-letter designations that may be encountered alternately herein.

Mutations can be made in an amino acid sequence or nucleic acid sequences encoding polypeptides and lysins herein, such as the lysin sequences presented or referred to herein, or active fragments or truncations thereof, such that a particular codon is changed to a codon encoding a different amino acid, an amino acid is substituted with another amino acid, or one or more amino acids are deleted. Such mutations are generally produced by making as few amino acid or nucleotide changes as possible. Substitution mutations of this kind can occur such that amino acids in the resulting protein are changed non-conservatively (e.g. by changing a codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another group) or conservatively (e.g. by changing a codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same group). Such conservative changes generally result in less variation in the structure and function of the resulting protein. Non-conservative changes are likely to alter the structure, activity or function of the resulting protein. The present invention is intended to include sequences with conservative changes that do not significantly alter the activity or binding properties of the resulting protein.

Thus, one of ordinary skill in the art can make amino acid changes or substitutions in the lysin polypeptide sequences based on examination of the sequences of the lysin polypeptides presented herein, particularly the lysin polypeptides having SH-3 type binding domains presented herein, and the knowledge of one of skill in the art and the public information available for other lysin polypeptides. Amino acid changes can be made to replace or replace one or more, one or a few, one or several, 1-5, 1-10, or such other number of amino acids in the lysin(s) sequence presented herein to generate a mutant or variant thereof. Such mutants or variants thereof can be predicted for function or tested for function or ability to kill bacteria, including Staphylococcus, Streptococcus, Listeria or Enterococcus, and/or have comparable activity to the lysin(s) presented herein. Thus, changes can be made to the sequences of the lysin polypeptides having SH-3 type binding domains of the invention, including PlySs2 (CF-301), Sal, LysK, etc., presented and referred to herein, for example by modifying the amino acid sequences presented or referred to herein, and mutants or variants having changes in the sequences can be tested using the assays and methods described and exemplified herein, eg, in the Examples. One skilled in the art can predict one or more, one or several amino acids suitable for substitution or substitution, including suitable conservative or non-conservative substitutions, and/or one or more amino acids not suitable for substitution or substitution, based on the domain structure of the lysin(s) herein.

In this regard, with exemplary reference to the PlySs2 (CF-301) lysin, but without limitation, it is pointed out that the PlySs2 (CF-301) polypeptide lysin comprises the N-terminal CHAP domain (cysteine-histidine amidohydrolase/peptidase) and the C-terminal SH3 type 5 domain shown herein. The domain is LNNV. . . starting with . . . a CHAP domain corresponding to the first amino acid sequence region ending with HYIT;
and RSYR. . . starting with . . . It is indicated by an SH-3 type domain corresponding to the second region ending in YVAT. Similarly, one can readily identify relevant N-terminal catalytic domains and/or C-terminal binding domains, particularly SH-3 type binding domains, in the lysin polypeptides referred to herein and used in the invention, including but not limited to Sal lysin, LysK lysin, lysostaphin, as well as Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. CHAP domains are contained in several previously characterized streptococcal and staphylococcal phage lysins. Thus, one skilled in the art can appropriately make and test substitutions or permutations to the CHAP and/or SH-3 domains of PlySs2 (CF-301). Sequence comparisons to the Genbank database can be performed using either or both CHAP and/or SH-3 domain sequences, or the PlySs2 (CF-301) lysin complete amino acid sequence, eg, to identify amino acids for substitution. CHAP domains include, for example, conserved cysteine and histidine amino acid sequences (underlined in the PlySs2 (CF-301) sequence herein). For example, it is reasonable to expect conserved cysteine and histidine residues to be maintained in mutants or variants of PlySs2 (CF-301) in order to maintain activity or potency. It is noteworthy that mutants or variants in which valine is replaced by alanine at valine amino acid 19 in the PlySs2(CF-301) sequence are active and capable of killing Gram-positive bacteria as and as effectively as the originally isolated and sequenced PlySs2(CF-301) lysin.

For example, PlySs2 (CF-301) lysin (SEQ ID NO:3) contains an N-terminal CHAP domain (LNNV...HYIT; amino acids 8-146) and a C-terminal SH3 domain (RSYR...YVAT; amino acids 162-228). Together, these two regions of domain sequence homology constitute 206 of the total 245 amino acids of the PlySs2 (CF-301) amino acid sequence (SEQ ID NO:3), accounting for 84% of the polypeptide sequence. Thus, most of the PlySs2 (CF-301) lysin amino acid sequence corresponds to domain homologous sequences. Structural/functional information is also available to those skilled in the art for each of the CHAP and SH3 domain sequences to contribute to the semi-rational design of variants. For example, Bateman and Rawlings (Bateman, A. and Rawlings ND (2003) Trends in Biochemical Sciences 28(5):234-237 "The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases") describe and identify CHAP domains in a number of polypeptides, demonstrate sequence variation in exemplary alignments, and identify essential invariant cysteine and histidine residues. are identified. This analysis and information was provided by Zou and Hou (Zou, Y. and Hou, C. (2010) Computational Biology and Chemistry 34:251-257 "Systematic analysis of an amidase domain CHAP in 12 Staphylococcus aureus genomes and 44 staphylococcal phage genomes") using sequence alignments, analysis of consensus secondary structure, sequence variation, and characterization of highly conserved residues and sequence signatures. extended in detailed phylogenetic analysis of CHAP domains across 50 bacterial and phage genomes, including Prokaryotic or bacterial SH3 domains have been described and characterized in published techniques, including structural characterization and identification of highly conserved and charged residues, hydrophobic residues, and the like. For example, Whisstock, JC and Lesk, AM ("SH3 domains in prokaryotes" Trends in Biochemical Sciences 24:132-133 (1999)) describe SH3 domain homology in bacteria and align amino acid sequences showing conserved residues and aspects. Following this literature, ponting et al ("eukaryotic Signaling Domain Homologogues in Arche and Bacteria. Ancient Ancient Ancient and Horizontal Gene Transfer" (199) 99) 289: 729-745) evaluates various domain homologs, including SH3, presenting an expanded array and alignment over numerous bacterial SH3B domain sequences, describing exemplary replacements, and emphasizing well-preserved amino acids.

The following are examples of various groups of amino acids:
Amino acids with non-polar R groups Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine Amino acids with uncharged polar R groups Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine
Aspartic acid, glutamic acid Basic amino acids (positively charged at pH 6.0)
Lysine, arginine, histidine (pH 6.0)

Another group can be those amino acids with a phenyl group:
phenylalanine, tryptophan, tyrosine

Another group can be by molecular weight (i.e., size of the R group):
Glycine 75 Alanine 89
Serine 105 Proline 115
Valine 117 Threonine 119
Cysteine 121 Leucine 131
Isoleucine 131 Asparagine 132
Aspartic acid 133 Glutamine 146
Lysine 146 Glutamic acid 147
Methionine 149 Histidine (at pH 6.0) 155
Phenylalanine 165 Arginine 174
Tyrosine 181 Tryptophan 204

Particularly preferred substitutions are as follows:
replacement of Arg by Lys (and vice versa) such that the positive charge can be maintained;
replacement of Asp with Glu (and vice versa) such that the negative charge can be maintained;
replacement of Thr with Ser such that a free —OH can be maintained, and
Replacement of Asn by Gln such that free _NH2 can be maintained.

Exemplary preferred conservative amino acid substitutions include any of the following:
leucine (L) for valine (V); threonine (T) for serine (S) and vice versa; valine (V) for isoleucine (I) and vice versa; glutamine (Q) for lysine (K) and vice versa; methionine (M) for isoleucine (I) and vice versa. substitution of asparagine (N) by serine (S) (and vice versa); substitution of leucine (L) for methionine (M) (and vice versa); substitution of lysine (L) for glutamic acid (E) (and vice versa); substitution of alanine (A) for serine (S) (and vice versa); ); replacement of isoleucine (I) with leucine (L) (and vice versa); replacement of arginine (R) with lysine (K) (and vice versa).

Amino acid substitutions can also be introduced to replace amino acids with particularly favorable properties. For example, a Cys can be introduced into a potential site for disulfide bridges with another Cys. His can be introduced as a particularly "catalytic" site (ie, His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro can be introduced for its particularly planar structure, which induces β-turns in the structure of the protein.

The polypeptides or epitopes described herein can be used to generate antibodies and can also be used to detect binding to molecules that recognize lysin or lysin protein. Another embodiment is a molecule such as an antibody or other specific binding agent that can be generated by, for example, routine immunization or the use of the epitope by a phage display approach, where the epitope can be used to screen a library for potential binders. Such molecules recognize one or more epitopes of a lysin protein or a nucleic acid encoding a lysin protein. An antibody that recognizes an epitope can be a monoclonal antibody, a humanized antibody or part of an antibody protein. It is desirable that a molecule that recognizes an epitope have a specific binding to that epitope that is at least 10 times stronger than it has to serum albumin. Specific binding can be measured as affinity (Km). More desirably, specific binding is at least 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ higher or even higher than for serum albumin under the same conditions.

In a desirable embodiment, the antibody or antibody fragment is a form useful for detecting the presence of lysin proteins, or alternatively, detecting the presence of bacteria susceptible to lysin proteins. In further embodiments, antibodies can be attached or otherwise associated with the lysin polypeptides of the invention, e.g., in chimeric or fusion proteins, and can serve to direct the lysin to a bacterial cell or strain of interest or target. Alternatively, the lysin polypeptide can serve to direct the antibody or act in conjunction with the antibody in completely or partially lysing the bacterial cell wall, e.g., the antibody can specifically bind to its epitope on or in the bacterium on or below the surface. For example, a lysin of the invention can attach to an anti-streptococcal antibody and direct the antibody to its epitope.

As will be appreciated by those skilled in the art, various forms and methods of antibody synthesis are known. Antibodies can be conjugated (covalently complexed) to reporter molecules or atoms such as fluorophores, enzymes that produce an optical signal, chemilumiphores, microparticles or radioactive atoms. Antibodies or antibody fragments can be synthesized in vivo after immunization of an animal, for example, antibodies or antibody fragments can be synthesized by cell culture after genetic recombination. Antibodies or antibody fragments can be produced by a combination of cellular synthesis and chemical modification.

An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific epitope. The term includes polyclonal, monoclonal and chimeric antibodies, which are described in more detail in US Pat. Nos. 4,816,397 and 4,816,567. The term "antibody" refers to an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also includes any polypeptide or protein having a binding domain that is, or is homologous to, an antibody binding domain. CDR-grafted antibodies are also contemplated by this term. An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific epitope. The term includes polyclonal, monoclonal and chimeric antibodies, which are described in more detail in US Pat. Nos. 4,816,397 and 4,816,567. The term "antibody(s)" includes wild-type immunoglobulin (Ig) molecules generally comprising four full-length polypeptide chains, two heavy (H) chains and two light (L) chains, or equivalent Ig homologues thereof, including full-length functional mutants, variants or derivatives thereof that retain the requisite epitope binding characteristics of an Ig molecule, including dual specific, bispecific and dual variable domain antibodies (e.g. camel family nanobodies). Immunoglobulin molecules may be of any class (eg IgG, IgE, IgM, IgD, IgA and IgY) or subclass (eg IgG1, IgG
2, IgG3, IgG4, IgA1 and IgA2). Any "antibody fragment" is also included within the meaning of the term "antibody."

An "antibody fragment" is defined as (i) a Fab fragment, which is a monovalent fragment consisting of variable light (VL), variable heavy (VH), constant light (CL), and constant heavy 1 (CH1) domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bridges at the hinge region; (iii) the heavy chain portion of a Fab (Fd) fragment, which consists of the VH and CH1 domains; (v) a domain antibody (dAb) fragment comprising a single variable domain (Ward, ES et al., Nature 341, 544-546 (1989)), (vi) a camelid antibody, (vii) an isolated complementarity determining region (CDR), (
viii) single-chain Fv fragments in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (ix) the VH and VL domains are expressed on a single polypeptide chain; Diabody (WO 94/13804, P. Holliger et al Proc. Natl. Acad. Sci. USA 90 6444-6448, (1993)), a bivalent, bispecific antibody that pairs a domain with a complementary domain on another chain, giving rise to two antigen-binding sites, using linkers that are too short to allow pairing between the two domains on the same chain, and (x) complementarity (xi) multivalent antibody fragments (scFv dimers, trimers and/or tetramers (Power and Hudson, J Immunol. Methods 242: 193-2049 (2000)), and (xii) heavy and/or light chains either alone or in any combination A molecule comprising at least one polypeptide chain that is not full-length, including other non-full-length portions of, or mutants, variants or derivatives thereof.

As antibodies can be modified in a number of ways, the term "antibody" shall be taken to encompass any specific binding member or substance having a binding domain with the required specificity. Thus, the term encompasses antibody fragments, derivatives, functional equivalents and homologues of antibodies, whether natural or wholly or partially synthetic, including any polypeptide comprising an immunoglobulin binding domain. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies is described in EP 0120694 and EP 0125023 and US Pat. Nos. 4,816,397 and 4,816,567.

An "antibody combining site" is that structural portion of an antibody molecule composed of the light or heavy and light chain variable and hypervariable regions that specifically binds antigen.

The term "antibody molecule" in its various grammatical forms, as used herein, contemplates both intact immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of immunoglobulin molecules that contain paratopes, including portions known in the art as Fab, Fab′, F(ab′) ₂ and F(v), which portions are preferred for use in the therapeutic methods described herein.

The term "monoclonal antibody" in its various grammatical forms refers to an antibody that has only one type of antibody combining site capable of immunoreacting with a specific antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. Thus, a monoclonal antibody can include an antibody molecule having multiple antibody combining sites, each immunospecific for a different antigen, eg, a bispecific (chimeric) monoclonal antibody.

The term "specific" can be used to refer to the situation in which one member of a specific binding pair does not exhibit significant binding to molecules other than its specific binding partner(s). The term also applies, for example, when an antigen-binding domain is specific for a particular epitope carried by multiple antigens, in which case a specific binding member carrying the antigen-binding domain can bind to different antigens carrying the epitope.

The term "comprising" is used generally to mean including, ie, allowing the presence of, one or more features or components.

The term "consisting essentially of" refers to a product, particularly a peptide sequence, of a defined number of residues not covalently attached to a larger product. However, it will be appreciated by those skilled in the art that in the case of the peptides of the invention herein, terminal chemical modifications adding protecting groups and the like, e.g. minor modifications to the N- or C-terminus of the peptide, such as C-terminal amidation, may be contemplated.

The term "isolated" refers to the state in which the lysin polypeptide(s) of the invention, or nucleic acids encoding such polypeptides, are in accordance with the invention. Polypeptides and nucleic acids are free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids found in their natural environment or the environment in which they are made (e.g., cell culture) when production is by recombinant DNA techniques performed in vitro or in vivo. Polypeptides and nucleic acids can be formulated with diluents or adjuvants and still isolated for practical purposes. For example, the polypeptides are commonly mixed with polymers or mucoadhesives or other carriers, or with pharmaceutically acceptable carriers or diluents when used diagnostically or therapeutically.

Nucleic Acids Nucleic acids capable of encoding a lysin polypeptide(s) of the invention are mentioned or presented herein or constitute aspects of the invention. Exemplary nucleic acid sequences in this context are polynucleotide sequences encoding any of the lysin polypeptides presented or referred to herein, and sequences that hybridize under stringent conditions to the DNA complementary sequences of the coding sequences. Additional variants of these sequences, including naturally occurring variants that may be obtained, and sequences of nucleic acids that hybridize thereto are also contemplated for use in making lysing enzymes according to this disclosure. A wide variety of isolated nucleic acid or cDNA sequences encoding phage-associated lytic enzymes, and subsequences that hybridize to such gene sequences, are useful for recombinant production of the lysin enzyme(s) or polypeptide(s) of the invention.

A "replicon" is any genetic element (eg, plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, ie, is capable of replication under its own control.

A "vector" is a replicon such as a plasmid, phage or cosmid to which another DNA segment can be attached so as to effect replication of the attached segment.

A "DNA molecule" refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) in either single-stranded form or double helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein according to the usual convention of producing sequences only in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having sequence homology to mRNA).

"Origin of replication" refers to a DNA sequence that participates in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (eg, mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA control sequences, such as promoters, enhancers, polyadenylation signals, terminators, etc., which provide for expression of a coding sequence in a host cell.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, a promoter sequence is bounded at its 3′ end by a transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is subsequently translated into the protein encoded by the coding sequence.

A "signal sequence" may be included before the coding sequence. This sequence encodes a signal peptide at the N-terminus of the polypeptide that communicates with the host cell and directs the polypeptide to the cell surface or secretes the polypeptide into the medium, which signal peptide is cleaved off by the host cell before the protein is released from the cell. Signal sequences can be found in association with a variety of proteins unique to prokaryotes and eukaryotes.

The term "oligonucleotide", as used herein with respect to probes of the invention, is defined as a molecule composed of two or more, preferably more than three, ribonucleotides. Its exact size depends on many factors, which in turn depend on the ultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whether naturally occurring as in a purified restriction digest or made synthetically, is capable of acting as an initiation point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of an inducing agent such as a nucleotide and a DNA polymerase, and at a suitable temperature and pH. A primer can be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

Primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, a primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer and the remainder of the primer sequence complementary to the strand. Alternatively, non-complementary bases or longer sequences may be incorporated into the primer, provided that the primer sequence has sufficient complementarity to hybridize with the sequence of the strand, thereby forming a template for synthesis of extension products.

As used herein, the terms "restriction endonuclease" and "restriction enzyme" each refer to bacterial enzymes that cut double-stranded DNA at or near specific nucleotide sequences.

A cell has been "transformed" by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. For example, in prokaryotes, yeast and mammalian cells, the transforming DNA can be maintained on episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome such that it is passed on to daughter cells by chromosomal replication. This stability is demonstrated by the ability of eukaryotic cells to establish cell lines or clones composed of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells arising from a single cell or common ancestor by mitosis. A "cell line" is a primary cell clone capable of stable growth in vitro for many generations.

Two DNA sequences are "substantially homologous" if at least about 75% (preferably at least about 80%, most preferably at least about 90% or 95%) of the nucleotides are identical over a defined length of the DNA sequence. Sequences that are substantially homologous can be identified by comparing sequences using standard software available in sequence databanks or in Southern hybridization experiments under, for example, stringent conditions, as defined for a particular system. Defining appropriate hybridization conditions is within the skill of those in the art. See, eg, Maniatis et al. (supra), DNA Cloning, Vols. I & II (supra), Nucleic Acid Hybridization (supra).

Many of the mutant DNA molecules contemplated herein include those produced by standard DNA mutagenesis methods such as M13 primer mutagenesis. Details of these techniques are presented in Sambrook et al. (1989) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, incorporated herein by reference. Using such techniques it is possible to generate variants slightly different from those disclosed. DNA molecules and nucleotide sequences that are derivatives of those specifically disclosed herein and that encode proteins that differ from those disclosed by deletion, addition or substitution of nucleotides, but still possess the functional properties of the lysin polypeptide(s) are contemplated by the present disclosure. Also included are small DNA molecules derived from the disclosed DNA molecules. Such small DNA molecules include oligonucleotides suitable for use as hybridization probes or polymerase chain reaction (PCR) primers. As such, these small DNA molecules comprise at least a segment of the lytic enzyme genetically encoded by the bacteriophage of Staphylococcus suis and, for PCR purposes, at least 10-15 nucleotide sequences of the gene, more preferably 15-30 nucleotide sequences. The DNA molecules and nucleotide sequences obtained above from the disclosed DNA molecules can also be defined as DNA sequences that hybridize to the disclosed DNA sequences or fragments thereof under stringent conditions.

Hybridization conditions corresponding to particular degrees of stringency will vary depending upon the nature of the hybridization method chosen and the composition and length of the hybridizing DNA used. Generally, the temperature of hybridization and the ionic strength (especially sodium ion concentration) of the hybridization buffer determine the stringency of hybridization. Calculations regarding hybridization conditions required to achieve particular degrees of stringency are discussed by Sambrook et al. (1989), In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., chapters 9 and 11, incorporated herein by reference.

An example of such a calculation is as follows. A hybridization experiment can be performed by hybridization of a DNA molecule (eg, a natural variation of a lytic enzyme genetically encoded by a bacteriophage specific for Bacillus anthracis) to a target DNA molecule. Target DNA is electrophoresed in an agarose gel, for example, by Southern blotting (Southern (1975). J. Mol. Biol. 98:503), a technique known in the art and described in Sambrook et al. (1989) In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, incorporated herein by reference.
It may be the corresponding cDNA transferred to a nitrocellulose membrane. Hybridization with isotope ^P32- labeled dCTP-labeled target probes is performed in a high ionic strength solution such as 6×SSC at 20° C.-25° C. below the melting temperature Tm (below). For such Southern hybridization experiments in which the target DNA molecules on the Southern blot contain 10 ng or more of DNA, hybridization is performed with 1 ng/ml to 2 ng/ml of radiolabeled probe (with a specific activity of 10 CPM/mug or more) for 6 to 8 hours. After hybridization, the nitrocellulose filters are washed to remove background hybridization. Wash conditions are as stringent as possible to remove background hybridization while retaining specific hybridization signal. The term "Tm" refers to the temperature above which a radiolabeled probe molecule will not hybridize to its target DNA molecule under prevailing ionic conditions. The Tm of such a hybrid molecule can be estimated from the following equation: _Tm = 81.5°C - 16.6 (log10 of sodium ion concentration) + 0.41 (% G+C) - 0.63 (% formamide) - (600/l), where l is the length of the hybrid in base pairs. This equation is valid for concentrations of sodium ions in the range of 0.01 M to 0.4 M and is less accurate for calculating Tm in solutions of higher sodium ion concentrations (Bolton and McCarthy (1962). Proc. Natl. Acad. Sci. USA 48:1390) (incorporated herein by reference). This equation is also valid for DNAs with G+C content within 30%-75% and applies to hybrids longer than 100 nucleotides in length. The behavior of oligonucleotide probes is described in detail in Chapter 11 of Sambrook et al. (1989), In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (incorporated herein by reference). The preferred exemplary conditions described herein are specifically contemplated for use in selecting mutations in lysis genes.

In preferred embodiments of the present disclosure, stringent conditions can be defined as conditions under which DNA molecules with more than 25% sequence variation (also called "mismatches") will not hybridize. In a more preferred embodiment, stringent conditions are conditions under which DNA molecules with more than 15% mismatches will not hybridize, and even more preferably stringent conditions are conditions under which DNA sequences with more than 10% mismatches will not hybridize. Stringent conditions are preferably conditions under which DNA sequences with more than 6% mismatches will not hybridize.

The degeneracy of the genetic code further broadens the scope of embodiments, as it allows for large variations in the nucleotide sequence of the DNA molecule while maintaining the amino acid sequence of the encoded protein. For example, a representative amino acid residue is alanine. Alanine can be encoded in the cDNA by the nucleotide codon triplet GCT. Due to the degeneracy of the genetic code, three other nucleotide codon triplets, GCT, GCC and GCA, also code for alanine. Thus, the nucleotide sequence of the gene can be changed to any of these three codons at this position without affecting the amino acid composition of the encoded protein or the properties of the protein. The genetic code and nucleotide codon variations for particular amino acids are known to those of skill in the art. Based on the degeneracy of the genetic code, variant DNA molecules can be obtained from the cDNA molecules disclosed herein using standard DNA mutagenesis techniques, as described above, or by synthesis of the DNA sequence. DNA sequences that do not hybridize to the disclosed cDNA sequences under stringent conditions due to sequence variation based on the degeneracy of the genetic code are included herein in the disclosure.

Thus, it is to be understood that DNA sequences encoding lysins of the invention, including PlySs2, which encode polypeptides having the same amino acid sequence as the sequences presented or referred to herein, but which are degenerate thereto, or degenerate to the exemplary nucleic acid sequences presented or referred to herein, are also within the scope of the present invention. "Degenerate to" means that different three-letter codons are used to designate particular amino acids. It is known in the art that the following codons can be used interchangeably to encode each specific amino acid:
Phenylalanine (Phe or F) UUU or UUC
Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or CUG
Isoleucine (Ile or I) AUU or AUC or AUA
Methionine (Met or M) AUG
Valine (Val or V) GUU or GUC or GUA or GUG
Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline (Pro or P) CCU or CCC or CCA or CCG
Threonine (Thr or T) ACU or ACC or ACA or ACG
Alanine (Ala or A) GCU or GCG or GCA or GCG
Tyrosine (Tyr or Y) UAU or UAC
Histidine (His or H) CAU or CAC
Glutamine (Gln or Q) CAA or CAG
Asparagine (Asn or N) AAU or AAC
Lysine (Lys or K) AAA or AAG
Aspartic acid (Asp or D) GAU or GAC
Glutamic acid (Glu or E) GAA or GAG
Cysteine (Cys or C) UGU or UGC
Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or AGG
Glycine (Gly or G) GGU or GGC or GGA or GGG
Tryptophan (Trp or W) UGG
stop codon UAA (ochre) or UAG (amber) or UGA (opal)

It should be understood that the codons specified above are for RNA sequences. The corresponding codon for DNA replaces U with T.

One skilled in the art will recognize that DNA mutagenesis methods described herein and known in the art can be used to generate a wide variety of DNA molecules that encode, by way of example, a bacteriophage lysin of the genus Streptococcus, yet retain the essential properties of the lytic polypeptides described and presented herein. Newly derived proteins can also be selected to obtain altered properties of the lytic polypeptide(s), as described more fully below. Such derivatives include those with amino acid sequence variations, including minor deletions, additions and substitutions.

Sites for introducing amino acid sequence mutations can be predetermined, but the mutations themselves need not be predetermined. For example, to optimize the performance of mutations at a given site, random mutagenesis can be performed at the target codon or region and the expressed protein variants screened for the optimal combination of desired activities. Techniques are known for making substitution mutations at predetermined sites in DNA having a known sequence as described above.

Amino acid substitutions are typically of single residues or may be of one or more, one or a few, 1, 2, 3, 4, 5, 6 or 7 residues; insertions are usually on the order of about 1-10 amino acid residues and deletions range from about 1-30 residues. Deletions or insertions may be of the single form, but are preferably made in adjacent pairs, ie, a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at the final construct. Obviously, mutations made in the protein-encoding DNA must not place the sequence out of reading frame and preferably do not create complementary regions that could result in secondary mRNA structure.

Substitutional variants are variants in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions can be made so as not to have a noticeable effect on the properties of the protein, or when it is desired to fine-tune the properties of the protein. Amino acids that can be substituted for the original amino acid in the protein and which are considered conservative substitutions are described above and recognized by those skilled in the art.

Substantial changes in function or immunological identity can be produced by choosing less conservative substitutions, for example by choosing residues that differ more significantly in their impact on maintaining (a) the structure of the polypeptide backbone in the replacement region, e.g., as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of side chains. Generally, substitutions expected to produce the greatest changes in protein properties are: (a) hydrophilic residues such as seryl or threonyl for hydrophobic residues such as leucyl, isoleucyl, phenylalanyl, valyl or alanyl (or hydrophilic residues for hydrophobic residues); (b) cysteine or proline for any other residue (or cysteine or proline for any other residue); (d) substitution of stydyl for electronegative residues such as glutamyl or aspartyl (or residues with electropositive side chains with electronegative residues); or (d) substitution of residues with bulky side chains such as phenylalanine with no side chains such as glycine (or residues with bulky side chains with residues without side chains).

The effect of these amino acid substitutions or deletions or additions can be assessed for derivatives or variants of the lytic polypeptide(s) by analyzing the ability of the derivative or variant protein to lyse or kill susceptible bacteria or to complement the sensitivity to DNA cross-linking agents displayed by phage in infected bacterial hosts. These assays can be performed by transfecting the above bacteria with a DNA molecule encoding the derivative or mutant protein or incubating the bacteria with proteins expressed from a host transfected with a DNA molecule encoding the derivative or mutant protein.

Sites for introducing amino acid sequence mutations can be predetermined, but the mutations themselves need not be predetermined. For example, to optimize the performance of mutations at a given site, random mutagenesis can be performed at the target codon or region and the expressed protein variants screened for the optimal combination of desired activities. Techniques are known for making substitution mutations at predetermined sites in DNA having a known sequence as described above.

Another feature of the invention is the expression of the DNA sequences disclosed herein. DNA sequences can be expressed by operably linking them to expression control sequences in a suitable expression vector and transforming a suitable unicellular host with the expression vector, as is known in the art. Such operative linking of the DNA sequences of the invention to expression control sequences, of course, involves providing the initiation codon ATG in correct reading frame upstream of the DNA sequence, if not already part of the DNA sequence. A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and plasmids such as known bacterial plasmids such as E. coli plasmids colE1, pCR1, pBR322, pMB9 and their derivatives, RP4; phage DNA such as numerous derivatives of phage λ such as NM989, and other phage DNA such as M13 and filamentous single-stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful for eukaryotic cells such as vectors useful for insect or mammalian cells. and vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences.

Any of a wide variety of expression control sequences (sequences that control the expression of DNA sequences operably linked thereto) can be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control region of the fd coat protein, the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g. Pho5), the yeast mating factor promoters, and prokaryotic or eukaryotic cells, or other sequences known to control the expression of those viral genes, as well as various combinations thereof.

A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts include fungi such as E. coli, strains of Pseudomonas, Bacillus, Streptomyces, yeast, and known eukaryotic and prokaryotic hosts such as CHO, R1.1, BW and LM cells in tissue culture, African green monkey kidney cells (e.g. COS 1, COS 7, BSC1, BSC40 and BMT10), insect cells (e.g. Sf9), and human and plant cells. can be included.

It is understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Also, not all hosts work equally well with the same expression system. However, one of ordinary skill in the art will be able to select, without undue experimentation, appropriate vectors, expression control sequences and hosts to achieve the desired expression without departing from the scope of the invention.

Libraries of fragments of polypeptide coding sequences can be used to generate mixed chain populations of polypeptides for screening and subsequent selection of variants. For example, the library of the code array fragment is that nicking occurs only about once per molecule per molecule, which is a cord sequence of the cord sequence that is interested in a two -strag arrangement of the cord sequence that is interested in it with a nucleuses, degenerate the double -stranded DNA, regenerates DNA, and derives the sense of the sense / antisense derived from different kicking products. It can be generated by forming a two -stranded DNA that can be obtained, removing a single -strag from a double chain that is re -formed by the processing of S1 nucleese, and rigating the obtained fragment library into an expression vector. By this method, expression libraries can be obtained that encode N-terminal and internal fragments of the protein of interest of various sizes.

Several techniques are known in the art for screening gene products of combinatorial libraries generated by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. The most widely used techniques amenable to high-throughput analysis for screening large gene libraries typically involve cloning the gene library into a replicable expression vector, transforming the resulting library of vectors into appropriate cells, and expressing the combinatorial genes under conditions where detection of the desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique to increase the frequency of functional mutants in libraries, can be used in conjunction with screening assays to identify protein variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815, Delgrave et al. (1993) Protein Engineering 6(3):327-331).

Compositions A therapeutic or pharmaceutical composition comprising the lytic enzyme(s)/polypeptide(s) of the invention and related methods of use and manufacture are provided according to the invention. Therapeutic or pharmaceutical compositions can comprise one or more lytic polypeptides, optionally native, truncated, chimeric or shuffled lytic enzymes, optionally in combination with carriers, vehicles, polypeptides, polynucleotides, holin protein(s), one or more antibiotics, or other ingredients such as suitable excipients, carriers or vehicles. The present invention relates to the lysins of the invention, particularly PlySs2 (CF-301), Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE, for use in killing, alleviating, decolonizing, prophylactic or treating Gram-positive bacteria, including bacteria of the genus Staphylococcus, including bacterial infections or related pathologies. Therapeutic or pharmaceutical compositions of lysins having SH3-type binding domains, including -1 lysins, are provided. The present invention relates to the lysins of the invention, particularly PlySs2 (CF-301), Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and A Therapeutic or pharmaceutical compositions of lysins having SH3-type binding domains, including LE-1 lysins, are provided. Compositions for topical or cutaneous application and general administration to the outside, including the skin or other external surfaces, are hereby contemplated and provided. PlySs2 (CF-301), S for killing, alleviating, decolonizing, prophylactic or treating Gram-positive bacteria, especially Streptococcus pyogenes, including Streptococcus pyogenes and antibiotic-resistant Staphylococcus aureus, Staphylococcus, Enterococcus or Listeria, including bacterial infections or related conditions
Presented herein are compositions comprising al lysins, LysK lysins, lysostaphin, phil lysins, LysH5 lysins, MV-L lysins, LysGH15 lysins and ALE-1 lysins, especially lysins with SH3-type binding domains including PlySs2 (CF-301), including domains, truncations or variants thereof.

The enzyme(s) or polypeptide(s) included in the therapeutic composition may be one or more or any combination of unmodified phage-associated lytic enzyme(s), truncated lytic polypeptides, mutant lytic polypeptide(s), and chimeric and/or shuffled lytic enzymes. In addition, different lytic polypeptide(s) genetically encoded by different phages for treatment of the same bacterium may be used. These lytic enzymes can also be "unmodified" lytic enzymes or polypeptides, truncated lytic polypeptide(s), mutant lytic polypeptide(s), and any combination of chimeric and shuffled lytic enzymes. The lytic enzyme(s)/polypeptide(s) in a therapeutic or pharmaceutical composition against Gram-positive bacteria including Streptococcus, Staphylococcus, Enterococcus and/or Listeria can be used alone or in combination with antibiotics or, if there are other invasive bacterial organisms to be treated, in combination with other phage-associated lytic enzymes specific for other bacteria to be targeted. Lytic enzymes, truncated enzymes, mutant enzymes, chimeric enzymes, chimeric polypeptides, fusion polypeptides, SH-3 binding domain-containing peptides or constructs, and/or shuffled lytic enzymes can be used with another therapeutic or antimicrobial peptide. Amounts of other therapeutic or antimicrobial peptides can also be varied. Various antibiotics may optionally be included in the therapeutic composition along with the enzyme(s) or polypeptide(s), with or without the presence of another therapeutic or antimicrobial peptide. More than one lytic enzyme or polypeptide may be included in the therapeutic composition.

Pharmaceutical compositions may comprise one or more modified lytic enzymes (including isozymes, analogs or variants thereof) produced by chemical synthesis or DNA recombinant methods. In particular, modified lytic proteins can be made by amino acid substitutions, deletions, truncations, chimerization, fusions, shuffling, or combinations thereof. A pharmaceutical composition may contain a combination of one or more naturally occurring lytic proteins and one or more truncated, mutant, chimeric or shuffled lytic proteins. Pharmaceutical compositions may contain peptides or peptide fragments of at least one lytic protein derived from the same or different bacterial species, optionally with the addition of one or more complementary agents, and a pharmaceutically acceptable carrier or diluent.

The present invention provides bacterial lysins, including lytic polypeptide variants such as PlySs2 (CF-301), Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin variants, particularly PlySs2 (CF-301) lysin polypeptide variants, which have bacterial killing activity. . In one aspect, the variant comprises or has an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% amino acid identity to any or one of SEQ ID NOs: 3-7, or any lysin polypeptide amino acid sequence presented herein, including any lysin polypeptide sequence referred to or presented herein by reference herein, particularly a reference lysin having an SH-3 type binding domain. The present invention includes SH-3 type lysin polypeptide truncation mutants, including PlySs2 (CF-301) lysin truncation mutants, which contain only a binding domain or only one catalytic or enzymatic domain and retain bacterial binding activity or Gram-positive antibacterial activity. The invention includes exemplary lysin cleavage mutants containing only one domain, eg, selected from binding domains, amidase domains and glucosaminidase domains. Truncation mutants, for example, lack an enzymatic domain, such as a glucosaminidase domain, such that the truncated lysin comprises and contains a replacement or alternative N-terminal enzymatic domain and a cell wall binding domain, particularly an SH3B-type binding domain.

The pharmaceutical composition may contain auxiliary substances including one or more antimicrobial agents and/or one or more conventional antibiotics. In order to accelerate the treatment of infections, the therapeutic agent may further comprise at least one auxiliary substance which can also enhance the bactericidal activity of the lytic enzymes. Antimicrobial agents primarily act by interfering with bacterial cell structure or function by inhibiting cell wall synthesis, inhibiting cell membrane function, and/or inhibiting metabolic functions including protein and DNA synthesis. Antibiotics can be broadly subdivided into those affecting cell wall peptidoglycan biosynthesis and those affecting DNA or protein synthesis in Gram-positive bacteria. Cell wall synthesis inhibitors, including penicillin and similar antibiotics, disrupt the hard outer cell wall such that relatively unsupported cells swell and eventually burst. Antibiotics that affect cell wall peptidoglycan biosynthesis include glycopeptides that inhibit peptidoglycan synthesis by preventing the incorporation of N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) peptide subunits into the peptidoglycan matrix. Available glycopeptides include vancomycin and teicoplanin, penicillins that act by inhibiting the formation of peptidoglycan crosslinks. The functional group of penicillin, the β-lactam moiety, binds to and inhibits DD-transpeptidase, which links peptidoglycan molecules in bacteria. Hydrolytic enzymes continue to degrade the cell wall, causing osmotic pressure-induced cell lysis or cell death. Common penicillins include oxacillin, ampicillin and cloxacillin, as well as polypeptides that prevent dephosphorylation of C ₅₅ -isoprenyl pyrophosphate, a molecule that carries peptidoglycan building blocks outside the plasma membrane. A polypeptide that affects the cell wall is bacitracin.

Auxiliary agents can be antibiotics such as erythromycin, clarithromycin, azithromycin, roxithromycin, other members of the macrolide family, penicillins, cephalosporins, and any combination thereof in amounts effective to synergistically enhance the therapeutic effect of the lytic enzymes. Virtually any other antibiotic can be used with modified and/or unmodified lytic enzymes. Similarly, other lytic enzymes may be included in the carrier to treat other bacterial infections. Antibiotic additives can be used in virtually all applications of enzymes in treating various diseases.

Also provided are compositions containing nucleic acid molecules capable of expressing in vivo effective amounts of a lytic polypeptide(s) or peptide fragment(s) of a lytic polypeptide, either alone or in combination with other nucleic acid molecules. Cell cultures containing these nucleic acid molecules, polynucleotides, and vectors carrying and expressing these molecules in vitro or in vivo are also provided.

A therapeutic or pharmaceutical composition may comprise the lytic polypeptide(s) in combination with various carriers to treat diseases caused by susceptible Gram-positive bacteria. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.かかる物質は、用いられる投与量及び濃度でレシピエントに対して非毒性であり、リン酸塩、クエン酸塩、コハク酸塩、酢酸及び他の有機酸、又はそれらの塩等の緩衝液；アスコルビン酸等の酸化防止剤；低分子量（約１０残基未満）ポリペプチド、例えばポリアルギニン又はトリペプチド；血清アルブミン、ゼラチン又は免疫グロブリン等のタンパク質；ポリビニルピロリドン等の親水性ポリマー；グリシン；グルタミン酸、アスパラギン酸、ヒスチジン又はアルギニン等のアミノ酸；単糖、二糖、及びセルロース若しくはその誘導体、グルコース、マンノース、トレハロース又はデキストリンを含む他の炭水化物；ＥＤＴＡ等のキレート剤；マンニトール又はソルビトール等の糖アルコール；ナトリウム等の対イオン；ポリソルベート、ポロキサマー又はポリエチレングリコール（ＰＥＧ）等の非イオン界面活性剤；及び／又は中性塩、例えばＮａＣｌ、ＫＣｌ、ＭｇＣｌ _２ 、ＣａＣｌ _２等を含む。 Glycerin or glycerol (1,2,3-propanetriol) is commercially available for pharmaceutical use. It can be diluted with sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluid, and used at a concentration of 0.1% to 100% (v/v), preferably 1.0% to 50%, more preferably about 20%. DMSO is an aprotic solvent with remarkable ability to enhance the permeation of many topically applied drugs. DMSO can be diluted with sterile water for injection, or sodium chloride injection, or other pharmaceutically acceptable aqueous injection fluids and used at concentrations of 0.1% to 100% (v/v). Carrier vehicles can also include Ringer's solution, buffered solutions, and dextrose solution, particularly when preparing intravenous solutions.

Any carrier for the lytic polypeptide(s) can be manufactured by conventional means. However, it is preferred that any mouthwash or similar type product be alcohol free to prevent denaturation of the polypeptide/enzyme. Similarly, if the lytic polypeptide(s) is included in a cough drop, gum, candy or lozenge during the manufacturing process, it should be included prior to hardening of the lozenge or candy, but after some cooling of the cough drop or candy to avoid thermal denaturation of the enzyme.

The lytic polypeptide(s) can be added to these substances in liquid form or in a lyophilized state, which solubilizes on contact with bodily fluids such as saliva. The polypeptide(s)/enzyme may be in micelles or liposomes.

The administration rate or amount of the modified or unmodified lytic enzyme/polypeptide(s) that is effective in treating an infection will depend, in part, on whether the lytic enzyme/polypeptide(s) is used therapeutically or prophylactically, the duration of the recipient's exposure to the infectious bacteria, the size and weight of the individual, and the like. The duration of use of the composition containing the enzyme/polypeptide(s) will also depend on whether the use is prophylactic (in which case the use may be hourly, daily or weekly over a short period of time) or therapeutic (in which case a more intensive schedule of composition use may be required so that use may continue for hours, days or weeks and/or daily or at specified time intervals within a day). Any dosage form used should provide the minimum number of units for the minimum amount of time. Concentrations of enzyme activity units that are believed to provide an effective amount or dosage of the enzyme may range from about 100 units/ml to about 500,000 units/ml (fluid in the wet or damp environment of the nasal passages and oral passages), optionally from about 100 units/ml to about 50,000 units/ml. More specifically, the time of exposure to the active enzyme/polypeptide(s) units can affect the desired concentration of active enzyme units per ml. Carriers classified as "long" or "slow" release carriers (e.g., certain nasal sprays or lozenges, etc.) may have or provide lower concentrations of active (enzyme) units per ml over a longer period of time, while "short" or "rapid" release carriers (e.g., mouthwashes, etc.) may have or provide higher concentrations of active (enzyme) units per ml for a shorter period of time. The amount of active units per ml and duration of exposure will depend on the nature of the infection, whether treatment is to be prophylactic or therapeutic, and other variables. There are situations where it may be necessary to have a much higher unit/ml dose or a lower unit/ml dose.

The lytic enzyme/polypeptide(s) shall be in an environment with a pH that allows activity of the lytic enzyme/polypeptide(s). For example, if a human individual is exposed to another human with a bacterial upper respiratory tract disorder, the lytic enzyme/polypeptide(s) will be present in the mucosal lining to prevent any colonization with the infecting bacteria. Prior to or at the time the modified lytic enzyme is subjected to a carrier system or oral delivery system, it is preferred that the enzyme be in a stabilizing buffer environment to maintain a pH range of from about 4.0 to about 9.0, more preferably from about 5.5 to about 7.5.

A stabilizing buffer can allow for optimal activity of the lysinase/polypeptide(s). The buffer may contain reducing reagents such as dithiothreitol. The stabilizing buffer may be or include a metal chelating reagent such as ethylenediaminetetraacetic acid disodium salt, or may contain a phosphate or citrate phosphate buffer, or any other buffer. The DNA encoding these and other phages can be modified to allow the recombinase to attack one cell wall at more than two locations, to cleave the cell walls of more than one bacterial species, to attack other bacteria, or any combination thereof. The types and number of modifications to enzymes produced by recombinant bacteriophage are numerous.

A mild surfactant may be included in the therapeutic or pharmaceutical composition in an effective amount to enhance the therapeutic effect of the lytic enzyme/polypeptide(s) that may be used in the composition. Suitable mild surfactants include, among others, esters of polyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxypolyethoxyethanol (Triton-X series), n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-decyl-β-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside. , and biologically occurring surfactants such as fatty acids, glycerides, monoglycerides, deoxycholate salts and esters of deoxycholic acid.

Preservatives may also be used in the present invention and preferably comprise from about 0.05% to 0.5% by weight of the total composition. The use of preservatives ensures that if the product becomes contaminated with microorganisms, the formulation will prevent or attenuate microbial growth. Some preservatives useful in the present invention include methylparaben, propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDM hydantoin, 3-iodo-2-propylbutylcarbamate, potassium sorbate, chlorhexidine digluconate, or combinations thereof.

Pharmaceutical agents for use in all embodiments of the present invention include anti-microbial agents, anti-inflammatory agents, anti-viral agents, local anesthetics, corticosteroids, destructive therapy agents, anti-fungal agents and anti-androgens. In the treatment of acne, active pharmaceutical agents that can be used include antimicrobial agents, especially those with anti-inflammatory properties such as dapsone, erythromycin, minocycline, tetracycline, clindamycin, and other antimicrobial agents. A preferred weight percentage for the antimicrobial agent is 0.5% to 10%.

Local anesthetics include tetracaine, tetracaine hydrochloride, lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride, dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride, butambenpicrate and pramoxine hydrochloride. A preferred concentration of local anesthetic is about 0.025% to 5% by weight of the total composition. Anesthetics such as benzocaine can also be used at preferred concentrations of about 2% to 25% by weight.

Corticosteroids that may be used include betamethasone dipropionate, fluocinolone actinide, betamethasone valerate, triamcinolone actinide, clobetasol propionate, desoxymethasone, diflorazone acetate, amcinonides, flurandrenolide, hydrocortisone valerate, hydrocortisone butyrate and desonide. , a concentration of about 0.01% to 1.0% by weight is recommended. Preferred concentrations of corticosteroids such as hydrocortisone or methylprednisolone acetate are from about 0.2% to about 5.0% by weight.

Additionally, the therapeutic composition may further comprise other enzymes such as the enzyme lysostaphin for treatment of any Staphylococcus aureus bacteria present with susceptible Gram-positive bacteria. Mucolytic peptides such as lysostaphin have been proposed to be effective in treating S. aureus infections in humans (Schaffner et al., Yale J. Biol. & Med., 39:230 (1967)). The gene product of Staphylococcus simulans, lysostaphin, exerts bacteriostatic and bactericidal effects on Staphylococcus aureus by enzymatically degrading polyglycine bridges in the cell wall (Browder et al., Res. Comm., 19: 393-400 (1965)). US Pat. No. 3,278,378 describes a fermentation process for making lysostaphin from the culture medium of S. staphylolyticus, later renamed Staphylococcus simulans. Other methods of making lysostaphin are further described in US Pat. Nos. 3,398,056 and 3,594,284. The lysostaphin gene was subsequently cloned and sequenced (Recsei et al., Proc. Natl. Acad. Sci. USA, 84: 1127-1131 (1987)). Recombinant mucolytic bactericidal proteins such as r-lysostaphin could potentially circumvent the problems associated with current antibiotic therapy because of their targeting specificity, low toxicity, and potential to reduce bioactive residues. Moreover, whereas most antibiotics require actively dividing cells to mediate their effects, lysostaphin is also active against non-dividing cells (Dixon et al., Yale J. Biology and Medicine, 41: 62-68 (1968)). Lysostaphin can be used in combination with modified lytic enzymes in the presence or absence of antibiotics. There is some additional importance in using both lysostaphin and lysinase in the same therapeutic agent. When humans have a bacterial infection, infection by one bacterial genus often weakens the human body or alters the body's flora, allowing other potentially pathogenic bacteria to infect the body. One of the bacteria that can co-infect the body is Staphylococcus aureus. The production of penicillinase by many strains of Staphylococcus aureus renders Staphylococci, Streptococci and other Gram-positive bacterial strains unkillable by standard antibiotics. As a result, the use of lysin and lysostaphin, optionally in combination with antibiotics, can be the most rapid and effective treatment of bacterial infections. A therapeutic composition may include mutanolysin and lysozyme.

Means of application of therapeutic compositions comprising lytic enzyme/polypeptide(s) include, but are not limited to, direct, indirect carriers and special means, or any combination of means. Direct application of the lytic enzyme/polypeptide(s) may be by any means suitable for direct contact of the polypeptide with the site of infection or bacterial colonization, such as application to the nasal area (e.g., nasal spray), dermal or skin application (e.g., topical ointment or formulation), suppository, tampon application, and the like. Nasal applications include, for example, nasal sprays, nasal drops, nasal ointments, nasal washes, intranasal injections, intranasal packings, bronchial sprays and inhalers, or indirectly by using throat lozenges, mouthrinses or gargles, or by using ointments applied to the nostrils or face, or any combination of these and similar application methods. Forms in which the lytic enzyme can be administered include, but are not limited to, lozenges, pastilles, candies, injectables, chewing gums, tablets, powders, sprays, liquids, ointments and aerosols.

When the natural and/or modified lytic enzyme(s)/polypeptide(s) are directly introduced using sprays, drops, ointments, washes, injections, packings and inhalers, the enzyme is preferably in a liquid or gel environment, with the liquid acting as the carrier. Although liquid delivery forms are preferred, the dry, anhydrous form of the modifying enzyme can be administered by inhalers and bronchial sprays.

A composition for treating a topical infection or contamination comprises according to the invention PlySs2 (CF-301), Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, in particular an effective amount of at least one lytic enzyme comprising PlySs2 (CF-301) and at least one carriers for delivering lytic enzymes to infected or contaminated skin, integuments or outer surfaces of mammals, humans, companion animals or livestock. Application modes for lytic enzymes include many different types and combinations of carriers including, but not limited to, aqueous liquids, alcoholic liquids, aqueous gels, lotions, ointments, non-aqueous liquid bases, mineral oil bases, blends of mineral oil and petrolatum, lanolin, liposomes, protein carriers such as serum albumin or gelatin, powdered cellulose, carmelose, and combinations thereof. Modes of delivery for carriers containing therapeutic agents include, but are not limited to, smears, sprays, time release patches, liquid absorbed wipes, and combinations thereof. The lytic enzyme can be applied directly to the dressing or in one of the other carriers. The bandage can be sold wet or dry (where the enzyme is in lyophilized form on the bandage). This method of application is most effective for treating infected skin. Topical composition carriers can include semisolid and gel vehicles including polymeric thickeners, water, preservatives, active surfactants or emulsifiers, antioxidants, sunscreens, and solvents or mixed solvent systems. US Pat. No. 5,863,560 (Osborne) discusses a number of different carrier combinations that can enhance skin exposure to the drug. Polymeric thickeners that can be used include those known to those skilled in the art such as hydrophilic and hydroalcoholic gelling agents commonly used in the cosmetic and pharmaceutical industries. CARBOPOL™ is one of many cross-linked acrylic acid polymers given the generic name carbomer. These polymers dissolve in water and form clear or slightly cloudy gels after neutralization with corrosive agents such as sodium hydroxide, potassium hydroxide, triethanolamine or other amine bases. KLUCEL™ is a cellulose polymer that disperses in water and forms a uniform gel after complete hydration. Other preferred gelling polymers include hydroxyethylcellulose, cellulose gum, MVE/MA decadiene crosspolymers, PVM/MA copolymers or combinations thereof.

Compositions containing the lytic enzyme/polypeptide(s) can be administered in the form of candies, chewing gums, lozenges, troches, tablets, powders, aerosols, liquids, liquid sprays or toothpastes for the prevention or treatment of bacterial infections associated with upper respiratory tract disease. The lozenge, tablet or gum to which the lytic enzyme/polypeptide(s) is added may contain sugar, corn syrup, various dyes, sugar-free sweeteners, flavors, optional binders, or combinations thereof. Similarly, any gum-based product may contain gum arabic, carnauba wax, citric acid, corn starch, food colors, flavors, sugar-free sweeteners, gelatin, glucose, glycerin, gum base, shellac, sodium saccharin, sugar, water, white wax, cellulose, other binders, and combinations thereof. Lozenges may further contain sucrose, corn starch, gum arabic, gum tragacanth, anethole, linseed, oleoresin, mineral oil and cellulose, other binders, and combinations thereof. Sugar substitutes can also be used in place of dextrose, sucrose or other sugars.

Compositions containing lytic enzymes or peptide fragments thereof can be directed to the mucosal lining where they kill resident and colonizing pathogenic bacteria. Mucosal linings include, for example, the upper and lower respiratory tract, the eye, oral cavity, nose, rectum, vagina, periodontal pockets, intestine and colon, as disclosed and described herein. Because of the natural elimination or cleansing mechanisms of mucosal tissue, conventional dosage forms are not retained at the site of application for any significant length of time.

It may be advantageous to administer a substance that exhibits adhesion to mucosal tissue together with one or more phage enzymes and other auxiliary substances over a period of time. Materials with controlled release capabilities are particularly desirable, and the use of sustained release mucoadhesives has received considerable attention. JR Robinson (US Pat. No. 4,615,697, incorporated herein by reference) provides a good review of various controlled release polymer compositions used for mucosal drug delivery. This patent describes a controlled release therapeutic composition comprising a bioadhesive material and an effective amount of a therapeutic agent. The bioadhesive material is a water-swellable but water-insoluble fibrous cross-linked carboxy-functional polymer containing (a) a plurality of repeating units, at least about 80% of which contain at least one carboxyl functional group, and (b) from about 0.05% to about 1.5% of a cross-linking agent that is substantially free of polyalkenyl polyethers. Robinson's polymers are water-swellable, but insoluble, crosslinked, not thermoplastic, and not as easy to formulate into various dosage forms with active agents as the copolymer systems of the present application. Micelles and multilamellar micelles can also be used to control the release of enzymes.

Other approaches are known using mucoadhesive agents that are a combination of hydrophilic and hydrophobic materials. Orahesive™ by E.R. Squibb & Co is an adhesive material that is a combination of pectin, gelatin and sodium carboxymethylcellulose in a sticky hydrocarbon polymer for adhesion to oral mucosa. However, such physical mixtures of hydrophilic and hydrophobic components eventually collapse. In contrast, the hydrophilic and hydrophobic domains in this application yield insoluble copolymers. US Pat. No. 4,948,580 (also incorporated herein by reference) describes a bioadhesive oral drug delivery system. The composition comprises a lyophilized polymer mixture formed from gelatin and poly(methyl vinyl ether/maleic anhydride) copolymer dispersed in an ointment base such as mineral oil containing dispersed polyethylene. U.S. Pat. No. 5,413,792 (incorporated herein by reference) discloses a pasty preparation comprising (A) a pasty base comprising a polyorganosiloxane and a water-soluble polymeric material, preferably present in a weight ratio of 3:6 to 6:3, and (B) an active ingredient. US Pat. No. 5,554,380 claims a solid or semi-solid, bioadhesive, orally ingestible drug delivery system containing a water-in-oil system having at least two phases. One phase comprises from about 25% to about 75% by volume of an internal hydrophilic phase and the other phase comprises from about 23% to about 75% by volume of an external hydrophobic phase, the external hydrophobic phase being composed of three components: (a) an emulsifier, (b) a glyceride ester and (c) a wax material. US Pat. No. 5,942,243, which is incorporated herein by reference, describes some representative release materials useful in administering antimicrobial agents.

A therapeutic or pharmaceutical composition may contain a polymeric mucoadhesive material comprising a graft copolymer comprising a hydrophilic backbone and a hydrophobic graft chain for controlled release of a bioactive agent. The graft copolymer is the reaction product of (1) a polystyrene macromonomer with ethylenically unsaturated functionality and (2) at least one hydrophilic acidic monomer with ethylenically unsaturated functionality. The graft chain consists essentially of polystyrene and a polymer backbone of hydrophilic monomeric moieties, some of which have acidic functional groups. The weight percent of polystyrene macromonomer in the graft copolymer is from about 1% to about 20%, the weight percent of total hydrophilic monomers in the graft copolymer is from 80% to 99%, wherein at least 10% of said total hydrophilic monomers are acidic, and said graft copolymer has an equilibrium water content of at least 90% when fully hydrated. Compositions containing the copolymer gradually hydrate at the site of application by adsorption of tissue fluids, yielding a very soft jelly-like mass that exhibits adhesion to mucosal surfaces. The composition, while adhering to the mucosal surface, provides sustained release of the pharmacologically active substance, which is absorbed by the mucosal tissue.

The compositions herein may optionally contain other polymeric substances such as poly(acrylic acid), poly(vinylpyrrolidone) and sodium carboxymethylcellulose plasticizers, as well as other pharmaceutically acceptable excipients, in amounts that do not adversely affect the mucoadhesive properties of the composition.

Dosage forms of the compositions of the present invention can be prepared by conventional methods. When intramuscular injection is the mode of administration of choice, it is preferable to use an isotonic formulation. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. A vasoconstrictor may be added to the formulation. Pharmaceutical preparations according to the present application are provided sterile and pyrogen-free.

The lytic enzyme/polypeptide(s) of the invention can also be administered by any pharmaceutically applicable or acceptable means, eg topically, orally or parenterally. For example, the lytic enzyme/polypeptide(s) can be administered intramuscularly, intrathecally, subdermally, subcutaneously or intravenously for the treatment of infections by Gram-positive bacteria. Isotonic formulations are preferably used when parenteral injection is the mode of administration of choice. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate-buffered saline are preferred. Stabilizers include gelatin and albumin. A vasoconstrictor may be added to the formulation. Pharmaceutical preparations according to the present application are provided sterile and pyrogen-free.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. Animal models are also used to achieve desired concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be chosen by the individual physician with regard to the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be considered include the severity of the patient's condition, age, weight and sex, diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivity, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

The effective rate or amount of parenterally administered lytic enzyme/polypeptide(s) and duration of treatment will depend, in part, on the severity of the infection, the weight of the patient, particularly the human, the duration of the recipient's exposure to the infectious bacteria, the number of square centimeters of skin or tissue infected, the depth of the infection, the severity of the infection, and a variety of numerous other variables. The compositions may be applied anywhere from once to several times a day and may be applied short or long term. Use can continue for several days or weeks. Any dosage form used should provide the minimum number of units for the minimum amount of time. The concentration of enzyme activity units believed to provide an effective amount or dosage of the enzyme can be selected as desired. The amount of activity units per ml and the duration of exposure will depend on the nature of the infection and the amount of contact the lytic enzyme(s)/polypeptide(s) are allowed to have by the carrier.

Methods and Assays The ability of the lysin polypeptide(s) of the invention to kill bacteria, indeed the remarkably broad spectrum of bacterial killing, provides a variety of methods based on the antibacterial efficacy of the polypeptide(s) of the invention. Thus, the present invention contemplates antimicrobial methods, including methods of killing Gram-positive bacteria, methods of reducing populations of Gram-positive bacteria, methods of treating or reducing bacterial infections, methods of treating human subjects exposed to pathogenic bacteria, and methods of treating human subjects at risk of such exposure. Susceptible bacteria include those from which the phage enzyme(s) of the invention are originally derived, and may also include various other strains of Streptococcus, Staphylococcus, Enterococcus and/or Listeria. Methods of prophylactic treatment of streptococcal, staphylococcal, enterococcal and/or listeria infections; methods of treating streptococcal, staphylococcal, enterococcal and/or listeria infections; methods of reducing streptococcal, staphylococcal, enterococcal and/or listeria populations or colonization; methods of treating lower respiratory tract infections; Also provided are methods of treating various conditions, including methods of treating , as well as methods of treating or preventing other local or systemic infections or conditions.

The lysin(s) of the present invention exhibit the ability to kill and effectiveness against bacteria from different species, e.g., bacteria from each of multiple Streptococcus or Staphylococcus species, Streptococcus, Staphylococcus, Enterococcus and/or Listeria, and across different species groups, such as bacteria from different orders. In particular, the lysin(s) of the present invention demonstrate the ability and efficacy to kill streptococcal and staphylococcal bacteria. In particular, the lysin(s) of the present invention demonstrate the ability and efficacy to kill Staphylococcus bacteria. PlySs2(CF-301) lysin exhibits the ability to kill bacteria from two different orders, in particular the order Bacillus and Lactobacilli, in vitro and in vivo. Thus, the present invention contemplates treatment, decolonization and/or decontamination of bacteria, cultures or infections, or where more than one Gram-positive bacteria is suspected or present. In particular, the present invention contemplates the treatment, decolonization and/or decontamination of suspected, present or possible bacteria, cultures or infections, or two or more Bacilli, two or more Lactobacilli, or at least one Bacillus and one Lactobacilli.

The invention may also be used to treat sepsis, particularly in humans. For treatment of septic infections such as pneumonia or bacterial meningitis, the therapeutic agent should be infused continuously intravenously into the bloodstream. Enzyme concentrations for the treatment of sepsis depend on the number of bacteria in the blood and blood volume.

Also provided is a method of treating a Streptococcus, Staphylococcus, Enterococcus and/or Listeria infection, colonization condition or population comprising treating the infection with a therapeutic agent comprising an effective amount of at least one lytic enzyme(s)/polypeptide(s) of the invention, particularly a lysin comprising an SH3-type binding domain, particularly PlySs2. at least one lytic enzyme(s)/polypeptide(s) of the invention, especially a lysin comprising an SH3-type binding domain, especially PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin or ALE-1 lysin, especially PlySs2 Also provided are methods of treating streptococcal infections, or methods of treating streptococcal and/or staphylococcal infections, colonization conditions or populations, comprising treating the infection with a therapeutic agent comprising an effective amount of (CF-301). In one aspect, lytic enzymes/polypeptides are produced or provided that are capable of lysing the cell walls of Streptococcus, Staphylococcus, Enterococcus and/or Listeria bacterial strains. In the method of the invention, the lysin polypeptide(s) of the invention, in particular the lysin comprising an SH3-type binding domain, such as in particular PlySs2 (CF-301), is useful and effective in prophylactic and therapeutic methods, especially against Gram-positive bacteria selected from Streptococcus, Staphylococcus, Enterococcus and/or Listeria infections, especially Streptococcus and/or Staphylococcus infections or bacterial colonization. Bacterial strains relevant as susceptible targets in the methods of the present invention include Staphylococcus aureus, Listeria monocytogenes, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo, Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS), Streptococcus sanguinis, Streptococcus golus Doni, Streptococcus dysgalactiae, Group G Streptococcus, Group E Streptococcus, Enterococcus faecalis and Streptococcus pneumoniae, and may be selected from these. In particular aspects, the bacterial strain is Staphylococcus aureus, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo, Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS), Streptococcus sanguinis, Streptococcus gordonii, Streptococcus dysgalactiae , group G streptococci, group E streptococci and pneumococci.

The present invention provides a method of treating or ameliorating a related infection or condition, including streptococci, including Streptococcus pyogenes, and/or staphylococci, including Staphylococcus aureus, particularly antibiotic-resistant Staphylococcus aureus, including MRSA, comprising contacting a bacterium, or a human subject infected with, exposed to, or suspected of or at risk of being exposed to, a specific bacterium with an amount of an isolated lysin polypeptide(s) of the invention effective to kill the specific bacterium. including methods of causing or administering the same. Thus, in particular lysins containing SH3-type binding domains, in particular PlySs2 (CF-301) lysins, Sal lysins, LysK lysins, lysostaphin, Phil lysins, LysH5 lysins, MV-L lysins, LysGH15 lysins and ALE-1 lysins, in particular PlySs2 (CF-301) (truncations thereof including such polypeptides presented and referred to herein). or variants) is contacted or administered to be effective in killing the bacteria of interest or otherwise to reduce or treat bacterial infection.

The term "agent" means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular, the term agent includes compounds such as test compounds, additional additional compound(s), or lysinase compounds.

The term "agonist" in its broadest sense refers to a ligand that stimulates the receptor to which it binds.

The term "assay" means any process used to measure a particular property of a compound. "Screening assay" means a process used to characterize or select compounds from a collection of compounds based on their activity.

The terms "prevent" or "prevention" refer to reducing the risk of having or developing a disease or disorder in a subject that may be exposed to or susceptible to a disease prior to disease onset (i.e., not developing at least one of the clinical symptoms of the disease).

The term "prophylaxis" is related to and encompassed by the term "prevention" and refers to measures or procedures whose purpose is to prevent, rather than treat or cure, disease. Non-limiting examples of preventive measures may include administration of vaccines, administration of low molecular weight heparin to hospitalized patients at risk of thrombosis, e.g., due to immobilization, and administration of antimalarial agents such as chloroquine prior to visiting geographic areas where malaria is common or at high risk of contracting malaria.

By "therapeutically effective amount" is meant that amount of a drug, compound, antimicrobial agent, antibody, polypeptide or pharmaceutical agent that elicits the biological or medical response in a subject sought by a physician or other clinician. In particular, with respect to Gram-positive bacterial infection and growth of Gram-positive bacteria, the term "effective amount" is intended to include an effective amount of a compound or agent that produces a biologically meaningful reduction in the amount of Gram-positive bacteria or the extent of infection, including having a bactericidal and/or bacteriostatic effect. The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, preferably reduce, by at least about 30%, more preferably at least 50%, and most preferably at least 90%, any clinically significant change in the growth or amount of infectious bacteria or other features of the pathology, such as fever or white blood cell count, that may accompany their presence and activity.

The term “treating” or “treatment” of any disease or infection refers, in one embodiment, to ameliorating the disease or infection (i.e., preventing the disease or the growth of infectious agents or bacteria, or reducing the onset, extent or severity of at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating a disease or infection physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. In further embodiments, "treating" or "treatment" relates to slowing disease progression or reducing infection.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions, such as acute gastric peristalsis, dizziness, etc., when administered to humans.

It is noted that the terms subject, patient or individual, in the context of therapeutic methods performed in vivo, or medical and clinical therapeutic methods according to the present application and claims, are intended to refer to humans.

The terms "gram-positive bacteria", "Gram-positive bacteria", "Gram-positive bacteria", and any variations not specifically mentioned may be used interchangeably herein and as used throughout this application and claims refer to Gram-positive bacteria that are known and/or identifiable by the presence of certain cell wall and/or cell membrane characteristics and/or staining with a Gram stain. Gram-positive bacteria are known and readily identifiable and may be selected from the genera Listeria, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium and Clostridium, including, but not limited to, any and all recognized or unrecognized species or strains thereof. In one aspect of the invention, the PlySs2 (CF-301) lysin-sensitive Gram-positive bacteria include bacteria selected from one or more of the genera Listeria, Staphylococcus, Streptococcus and Enterococcus, particularly Streptococcus and Staphylococcus bacteria. LysK and Sal1 lysin sensitive bacteria include Staphylococcus bacteria.

The term "sterilization" refers to being able to kill bacterial cells.

The term "bacteriostatic" refers to the ability to inhibit bacterial growth, including inhibition of proliferating bacterial cells.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce allergic or similar adverse reactions, such as acute gastric peristalsis, dizziness, etc., when administered to humans.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, preferably reduce by at least about 30%, more preferably at least 50%, and most preferably at least 90%, the S-phase activity of the target cell mass or other clinically significant changes in the pathology that may be associated with its presence and activity, such as elevated blood pressure, fever, or white blood cell count.

One method of treating systemic or tissue bacterial infections caused by streptococcal or staphylococcal bacteria is the use of one or more of the lysin polypeptides of the invention, particularly PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin, particularly PlySs2 ( CF-301), including fusions, chimeras, truncations or variants thereof, including such polypeptides presented herein, and a suitable carrier. A number of other different methods can be used to introduce the lytic enzyme(s)/polypeptide(s). These methods include intravenous, intramuscular, subcutaneous, intrathecal and subdermal introduction of the lytic enzyme(s)/polypeptide(s). Those skilled in the art, including medical practitioners, will be able to assess and appreciate the most appropriate mode of administration or means given the nature and extent of the bacterial condition and the strain or species of bacteria involved or suspected. For example, the intrathecal use and administration of one or more lytic polypeptides is most beneficial for the treatment of bacterial meningitis.

Infections can also be treated by injecting into the infected tissue of a human patient a therapeutic agent comprising the appropriate lytic enzyme(s)/polypeptide(s) and a carrier for the enzyme. The carrier may consist of distilled water, saline, albumin, serum or any combination thereof. More specifically, infusion or injectable solutions can be prepared in a conventional manner with the addition of preservatives such as p-hydroxybenzoate or stabilizers such as alkali metal salts of ethylene-diaminetetraacetic acid, which can then be transferred to infusion containers, injection vials or ampoules. Alternatively, compounds for injection can be lyophilized, with or without other ingredients, and solubilized in buffered solutions or distilled water as needed at the time of use. Non-aqueous vehicles such as fixed oils, liposomes and ethyl oleate are also useful herein. Other phage-associated lytic enzymes may be included in the composition along with the holin protein.

Lysins especially containing SH3-type binding domains as a prophylactic treatment to eliminate or reduce carriage of susceptible bacteria and to prevent becoming ill in humans exposed to other humans with symptoms of infection, or as therapeutic treatments for infections in humans already ill, especially the PlySs2 (CF-301) lysins, Sal lysins, LysK lysins, Lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin exemplified herein. , LysGH15 lysin and ALE-1 lysin, and ALE-1 lysin, for example PlySs2 (CF-301). Similarly, the lytic enzyme(s)/polypeptide(s) can be used to treat, for example, lower respiratory tract disease, particularly through the use of bronchial sprays or intravenous administration of enzymes. For example, lytic enzymes can be used for prophylactic and curative treatment of ocular infections such as conjunctivitis. Methods of treatment include administering eye drops or washes comprising an effective amount of at least one lytic polypeptide of the present invention and a carrier that can be safely applied to the eye, the carrier containing a lytic enzyme. Eye drops or washes are preferably in the form of isotonic solutions. The pH of the solution shall be adjusted so as not to cause possible infection by other organisms and possibly inflammation of the eye leading to damage to the eye. The pH range should be the same as for other lytic enzymes, but the optimum pH is the range demonstrated and presented herein. Similarly, buffers of the type described above for other lytic enzymes shall also be used. Other antibiotics suitable for use in eye drops can be added to the composition containing the enzyme. Bactericides and bacteriostatic compounds may be added. The concentration of enzyme(s) in solution can range from about 100 units/ml to about 500,000 units/ml, with more preferred ranges from about 100 units/ml to about 5000 units/ml and from about 100 units/ml to about 50000 units/ml. Concentrations can be higher or lower than the ranges given.

The lytic polypeptide(s) of the invention can also be used in contact lens solutions for soaking and washing contact lenses. This solution is usually an isotonic solution and can contain, in addition to enzymes, sodium chloride, mannitol and other sugar alcohols, borates, preservatives and the like. The lytic enzymes/polypeptides of the invention can also be administered to the patient's ear. Thus, for example, the lytic polypeptide(s) of the invention can be used to treat ear infections caused by, for example, Streptococcus pneumoniae. Otitis media is an inflammation of the middle ear characterized by symptoms such as ear pain, hearing loss and fever. One of the main causes of these symptoms is the accumulation of fluid (exudate) in the middle ear. Complications include permanent hearing loss, tympanic membrane perforation, pseudocholesteatoma, mastoiditis and adhesive otitis media. Children who develop otitis media within the first few years of life are at risk of recurrent acute or chronic disease. One of the major causes of otitis media is Streptococcus pneumoniae. The lytic enzyme(s)/polypeptide(s) can be applied to the infected ear by delivering the enzyme(s) in a suitable carrier to the ear canal. Carriers may comprise sterile aqueous or oily solutions or suspensions. The lytic enzyme(s) may be added to the carrier which may also contain suitable preservatives, preferably surfactants. Bactericidal and fungicidal agents preferably included in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for preparing oily solutions include glycerol, dilute alcohol and propylene glycol. Additionally, any number of other eardrop carriers may be used. The concentrations and preservatives used to treat otitis media and other similar ear infections are the same as those discussed for eye infections, and the carriers in which the enzyme is placed are similar or identical to those for treatment of eye infections. Additionally, carriers typically include vitamins, minerals, carbohydrates, sugars, amino acids, proteinaceous substances, fatty acids, phospholipids, antioxidants, phenolic compounds, isotonic solutions, oil solutions, oil suspensions and combinations thereof.

The diagnostic, prophylactic and therapeutic possibilities and uses provided by the recognition and isolation of the lysin polypeptide(s) of the invention derive from the fact that the polypeptides of the invention produce a direct and specific effect (e.g., killing) in susceptible bacteria. Thus, the polypeptides of the invention can be used to eliminate, characterize or identify relevant susceptible bacteria.

Thus, the diagnostic methods of the invention may involve testing a cell sample or medium with an assay comprising an effective amount of one or more lysin polypeptides and a means of characterizing one or more cells in the sample or determining whether cell lysis has occurred or has occurred, for the purpose of determining whether it contains susceptible bacteria or whether the bacteria in the sample or medium are susceptible. Patients who may benefit from this method include those with undetermined infections, recognized bacterial infections, or those suspected of being exposed to or carrying particular bacteria. Fluids, foodstuffs, medical devices, compositions or other such samples that come in contact with a subject or patient can be tested for susceptible bacteria or cleared of relevant bacteria. In one such aspect, a fluid, food product, medical device, composition or other such sample can be sterilized or otherwise treated by incubation with or exposure to one or more lytic polypeptides of the invention to eliminate or remove any potentially associated bacteria.

All procedures and their applications are well known to those skilled in the art and can therefore be used within the scope of the present invention. In one example, a lytic polypeptide(s) of the invention is complexed or otherwise bound or associated with relevant or susceptible bacteria in a sample and one member of the complex is labeled with a detectable label. The fact that a complex is formed, and, if desired, the amount thereof, can be determined by known methods applicable to detection of labels. The most commonly used labels for these studies are radioactive elements, enzymes, chemicals that fluoresce when exposed to ultraviolet light, and the like. Many fluorescent substances are known and can be used as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA Blue and Lucifer Yellow. Radiolabels can be detected by any of the currently available counting procedures. Preferred isotopes may be selected from ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I and ¹⁸⁶ Re. Enzymatic labels are likewise useful, and can be detected by any of the presently used colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric methods. Enzymes are conjugated to selected particles by reaction with cross-linking molecules such as carbodiimides, diisocyanates, glutaraldehyde, and the like. Many enzymes are known and can be used that can be used in these procedures. Peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase are preferred. US Pat. Nos. 3,654,090, 3,850,752 and 4,016,043 are cited by way of example for their disclosure of alternative labeling materials and methods.

PlySs2 (CF-301) lysin exhibits activity and ability to kill many different strains and species of Gram-positive bacteria, including Staphylococcus, Streptococcus, Listeria, or Enterococcus. Of particular importance, PlySs2(CF-301) is active in killing Staphylococcus aureus, particularly strains of the genus Staphylococcus, including both antibiotic-sensitive strains and different antibiotic-resistant strains. PlySs2 (CF-301) is also active in killing streptococcal strains, showing particularly effective killing against group A and group B streptococcal strains. The potency of PlySs2 (CF-301) lysin against bacteria, based on log killing assessments with isolates in vitro, is shown in Table 1 below. The susceptible bacteria presented herein can be used in the improved BMD method of the invention for determining and comparing MIC values.

The invention can be better understood by reference to the following non-limiting examples, which are presented as illustrations of the invention. The following examples are presented to more fully illustrate the preferred embodiments of the invention, but are not to be construed as limiting the broad scope of the invention in any way.

Example 1
Bacteriophage-derived lysins are cell wall hydrolases that offer new therapeutic options to combat the emergence and spread of drug-resistant bacterial pathogens. As a purified recombinant protein, lysins exhibit rapid species-specific lytic efficacy, anti-biofilm activity, low propensity for resistance, and significant synergy with antibiotics. In an effort to investigate the therapeutic potential of lysins, we discovered a strong "potentiating effect" that human blood matrix exhibits on the antistaphylococcal activity of the lysin PlySs2 (CF-301). The activity of PlySs2 (CF-301) in whole blood, serum and plasma results in a >100-fold reduction in minimal sterilizing concentration (time-kill assay) and a >32-fold decrease in minimal inhibitory concentration (microdilution assay) compared to activity in conventional media (cation-adjusted Mueller Hinton broth (caMHB)) across various S. aureus strains. Potentiating effects are further enhanced by synergistic combinations of PlySs2 (CF-301) with either daptomycin or vancomycin. Thus, PlySs2(CF-301) showed substantially greater potency (32-fold to >100-fold) in human blood compared to caMHB in standard microbiological test formats (e.g., MIC, checkerboard and time-kill assays).

Potentiating activity with other antistaphylococcal lysins was also noted. Human, rabbit, horse and dog sera showed comparable enhancing effects on PlySs2 (CF-301) activity, but this effect was moderate in rat and calf and absent in mouse sera. It provides additional evidence that the potentiation mechanism involves synergy with at least two blood components, lysozyme and serum albumin, which can be added in multiple assay formats to generalize blood effects. Finally, predictions based on ex vivo studies were confirmed in vivo by a study showing a superior efficacy profile for PlySs2(CF-301) (in addition to daptomycin) compared to rats in the treatment of infective endocarditis in rabbits. These findings were validated in vivo using established rabbit and rat models of S. aureus-infected endocarditis (IE) by demonstrating comparable efficacy at 111-fold lower doses in rabbit versus rat models. Overall, these findings suggest that favorable synergistic interactions between PlySs2 (CF-301) and serum proteins serve to promote bactericidal activity and are expected to have important therapeutic implications.

The emergence and spread of drug-resistant and multidrug-resistant bacteria has created a need for new alternative or adjunctive therapies to conventional antibiotics. One of the promising approaches currently under development is based on the use of recombinantly produced bacteriophage-derived lysins (cell wall hydrolases) to kill Gram-positive bacterial pathogens (1, 2). Lysins are antimicrobial enzymes that offer novel alternatives to traditional antibiotics. Lysins are proteins encoded by bacteriophages and used to kill bacteria in their natural setting. There are approximately ¹⁰ phages in the biosphere, and phages kill approximately one-third of all bacteria daily using the lysin protein family as their primary means of killing host bacteria (Hatful GF (2015) J Virol 89(16):8107-8110). Purified lysin exhibits a phenomenon called "lysis from without" (Fischetti VA et al (20016) Nature Biotechnology 24:1508-1511) and is suitable for synthetic recombinant production. Purified lysin exhibits a strong lytic effect via cell wall hydrolysis upon contact. Lysin polypeptides are typically 20-30 kDa proteins.

CF-301, PlySs2 lysin, also designated as PlySs2 (CF-301), is an antistaphylococcal lysin and the first lysin class agent to enter Phase 2 clinical development in the United States for the treatment of bacteremia, including endocarditis caused by Staphylococcus aureus (3). PlySs2 (CF-301) was originally derived from a prophage carried by S. suis in pigs. PlySs2 (CF-301) lysin was demonstrated to kill various strains of clinically significant Gram-positive bacteria, including antibiotic-resistant strains such as methicillin- and vancomycin-resistant and susceptible strains of Staphylococcus aureus (MRSA, MSSA, VRSA, VISA), daptomycin-resistant Staphylococcus aureus (DRSA), and linezolid-resistant Staphylococcus aureus (LRSA). PlySs2 (CF-301) has relatively broad but limited species-killing activity, being able to kill multiple bacterial species, particularly Gram-positive bacteria including strains of Staphylococcus, Streptococcus, Enterococcus and Listeria, but is inactive against bacteria in the natural gut flora.

Clinical grade PlySs2/CF-301 has been recombinantly produced in E. coli and is active over a wide pH (pH 6-9.7) and temperature (16°C-55°C) range (Gilmer et al (2013) Antimicrob Agents Chemother 57:2743-2750, Scuch et al (2014) J Infect Dis 209:1469-78). blood, serum, plasma, saliva,
It is active in a variety of human matrices including synovial fluid, pulmonary surfactant and bronchial lavage fluid. The amino acid sequence and structure of PlySs2 (CF-301) is presented herein above.

PlySs2 (CF-301) targets the cell walls of susceptible bacteria, including Staphylococcus aureus. It is a cysteine-histidine aminopeptidase that targets the D-Ala-L-Gly bond in cell wall peptidoglycan and cleaves between the cell wall D-alanine (stem peptide) and L-glycine (bridge). Lysis is rapid. PlySs2 (CF-301) has limited species specificity and kills antibiotic-resistant bacteria including MSSA, MRSA, VRSA, DRSA and LRSA, and bacteria resistant to methicillin, vancomycin, daptomycin, linezolid antibiotics (Schuch R et al (2014) J Infect Dis 209:1469-1478). Killing is rapid and robust with a low resistance profile to lytic peptides. PlySs2 (CF-301) eradicates biofilms and kills persistent bacteria. Effectiveness against biofilms is described in WO2013/170022 and US Pat. No. 9,499,594, incorporated herein by reference. Synergy has been observed with antibiotics, including those described in WO2013/170015 (incorporated herein by reference).

Hallmark features of PlySs2 (CF-301) include (i) potent, targeted and rapid lytic effect against a wide range of S. aureus isolates, (ii) anti-biofilm activity, (iii) low propensity for resistance, and (iv) synergy with conventional antibiotics (4, 5). Although PlySs2 (CF-301), in fact many additional lysins described in the literature, are highly effective lysing agents under standard in vitro test media conditions and are highly effective in a variety of animal models of invasive and topical infection, the activity of lysins in complex human physiological fluids such as whole blood, plasma and serum is poorly understood.

In the preclinical stages of antimicrobial drug development, initial evaluation of therapeutic potential is based on assays performed using artificial media to determine the minimum inhibitory concentration (MIC) and assess time-dependent killing in a time-killing assay format (6). However, it is also known that in vivo efficacy (particularly for systemically delivered drugs) is affected by multifactor interactions with constituents of the human body, particularly those of the blood. For many antibiotic classes, including β-lactams, quinolones and cyclic lipopeptides, the correlation (7-11) between plasma protein binding and decreased efficacy (up to 10-fold) to account for dosing regimens is very well studied. For example, binding to blood components such as albumin, α ₁ -acid glycoproteins, lipoproteins, α-, β- and γ-globulins, and red blood cells can reduce the amount of free active drug. Conversely, a growing number of studies also demonstrate the ability of human blood matrix to enhance (up to 16-fold) the antibacterial activity of both antibiotics (12, 13) and antimicrobial peptides (13-17). The ability of serum to enhance antimicrobial activity, for example, has been attributed to multiple factors ranging from effects on growth rate (7, 18) to synergistic interactions with humoral effectors of the innate and adaptive immune system (13, 15, 16, 19). The development of agents that may have both potent (endogenous) antimicrobial activity and the ability to augment pre-existing antimicrobial activity in the human blood environment offers very attractive therapeutic options as stand-alone agents or in combination with antibiotics.

Given the clinical program for systemic use of PlySs2(CF-301), human blood is the optimal testing matrix for PlySs2(CF-301) activity. The current investigation used an ex vivo screen for lysin PlySs2 (CF-301) activity against various S. aureus isolates in the context of human blood matrix. The antistaphylococcal activity of PlySs2 (CF-301) as a single agent and in combination with antibiotics was found to be consistently more effective in blood matrix compared to artificial media. Furthermore, human lysozyme and serum albumin each showed strong synergy with PlySs2 (CF-301), explaining many of the blood-enhancing effects observed in vitro. As an in vivo proof-of-concept, the efficacy of PlySs2 (CF-301) was tested in infectious endocarditis models using both rabbits (with human-equivalent hematologic effects) and rats (with moderate hematologic effects). In vivo studies demonstrated that approximately 50-fold higher doses were required for bactericidal effect in rats compared to rabbits. Overall, our results demonstrate that the antimicrobial activity of PlySs2 (CF-301) is enhanced in human blood by synergy with at least two blood components. The ability of PlySs2 (CF-301) to exhibit synergy with blood factors and antibiotics in complex human body fluids is a very important attribute of novel antimicrobials currently in clinical development.

Results Time-Dependent Killing in Human Blood Matrix A time-killing assay was used to assess the time-dependent bactericidal activity of PlySs2 (CF-301) against methicillin-resistant Staphylococcus aureus (MRSA) strain MW2 over various concentrations. Methodology published by the Clinical and Laboratory Standards Institute (CLSI) (CLSI document M07-A9 (Methods for dilutional antimicrobial sensitivity tests for bacteria that grow aerobically. Volume 32 (Wayne [PA]: Clinical and Laboratory Standards Institute [US], 2012), in which the standard test medium (i.e., Mueller Hinton Broth (MHB)) was replaced with human whole (heparinized) blood, serum, or plasma. In the combined time-kill curve, PlySs2 (CF-301) was rapidly bactericidal (greater than 3-log 10 (CFU/mL) reduction) and sterile (by 24 h post-treatment) at concentrations down to 3.2 μg/mL in all human blood matrices (Figures 1A, 1B and data not shown). The sterilizing concentration was 320 μg/mL (Fig. 1C).The 100-fold difference in sterilizing activity observed for MW2 was also observed for three additional MRSA strains, three methicillin-susceptible Staphylococcus aureus strains (MSSA) and one S. pyogenes strain (Fig. 2).In addition to PlySs2 (CF-301), a second lysin-like enzyme, lysostaphin (20), was also tested, although Pl Like ySs2(CF-301), it showed a 100-fold reduction in the concentration required for sterilization in blood compared to MHB (Figures 3A and 3B).As a control, vancomycin was also tested and showed slightly reduced potency in blood than in MHB (Figures 3C and 3D).

Time-kill experiments were also performed with PlySs2 (CF-301) to test the extent of the hematologic effect in different species (compared to human matrix and MHB). The activity of PlySs2 (CF-301) in mouse serum was similar to MHB, with sterilizing concentrations greater than 320 μg/mL (Fig. 1D). A moderate effect was observed with calf and rat sera at a sterile concentration of 32 μg/mL (FIGS. 4A and 4B). Human-like blood effects were observed in rabbit and dog sera at a sterile concentration of 3.2 μg/mL (FIGS. 4C and 4D). The time-kill results are consistent with the following order of blood-related enhancement: human = rabbit = dog > rat = calf > MHB > mouse. Previous studies have shown that horse serum can replace human serum in time-killing assays, especially with the addition of reducing agents such as DTT. International Application No. PCT US2017/32344 filed May 12, 2017 based on U.S. Patent Application No. 62/335,129 filed May 12, 2016, which is incorporated herein by reference. Horse sera show a similar enhancement, thus human=rat=dog=horse.

Minimum Inhibitory Concentration in Human Blood Matrix Minimum Inhibitory Concentration (MIC) provides a quantitative measure of antimicrobial activity in a static system at a fixed endpoint of 18 hours. PlySs2 (CF-301) activity was assessed in either standard test media (ie, MHB) or human serum against a variety of 171 clinical S. aureus isolates using the CLSI method of determining MICs by broth microdilution. PlySs2(CF-301) showed enhanced potency in human serum compared to MHB for all strains tested, including 74 MSSA, 75 MSSA, and additional vancomycin-, linezolid-, and daptomycin-resistant strains (Table 1). Overall, there was a 32-fold reduction in the PlySs2 (CF-301) concentration required to inhibit growth (MIC ₉₀ ) for 90% of the isolates tested in each group. Interestingly, both the antistaphylococcal lysin Sal1 and the lysin-like protein lysostaphin also showed a significant 32-fold reduction in MIC when tested in blood matrix (Table 2). However, the lysin ClyS showed only a slight 2-fold shift.

To understand how variation in the source of human blood-derived matrix can affect activity, the MICs of PlySs2 (CF-301) were determined using an array of 62 different pooled and individual human donor samples of whole blood, serum and plasma from different commercial sources varying in age, sex, blood type and type of anticoagulant used. For the whole blood (n=13), serum (n=33) and plasma (n=15) samples tested, MIC ₉₀ values for PlySs2 (CF-301) of 1 μg/mL ranging from 0.5 μg/mL to 2 μg/mL were observed (Table 3). The histograms showing the frequency of MICs for each of the various matrices show that the activities in whole blood and serum are comparable, but the activity in plasma differs by about 1-log2 (dilution) (Figure 5). Overall, the enhancing effect was unaffected by variations in individual or pooled donor age, sex and blood type, or the use of sodium citrate or sodium heparin as anticoagulants (Table 3). This effect was also observed in complement inactivated media, complement preserved media, and was comparable in matched sets of at least three different human blood, serum and plasma fractions, but not in delipidated serum.

The effect of sample variation on PlySs2 (CF-301) activity in sera of various animal species was also tested. The MIC range for mouse samples (n=10) was 32 μg/mL to 64 μg/mL, while for rats (n=5) the range was 8 μg/ml to 16 μg/ml, for dogs (n=8) was 0.5 μg/ml to 1 μg/ml, and for rabbits (n=2) was 1 μg/mL (Table 4). The order observed here, with rabbits and dogs superior to rats and mice, is identical to that observed in the time-killing studies described above.

PlySs2 (CF-301) Synergizes with Antibiotics in Human Blood Matrix The synergy of PlySs2 (CF-301) with either lysostaphin, daptomycin or vancomycin in the context of human serum was assessed using two different methods. The first method was a time-kill assay, the preferred technique for testing synergistic antimicrobial activity in vitro. Sub-MIC amounts of daptomycin and PlySs2 (CF-301) were tested individually and found to have minimal bactericidal effect against S. aureus strain MW2 (FIGS. 6A-6C). However, when the same amount of each agent was combined, substantially more killing was observed (2-log10
reduction of more than CFU/mL). A highly synergistic interaction was also observed for sub-MIC PlySs2 (CF-301) in combination with either lysostaphin (FIG. 6D) or vancomycin (data not shown).

In a time-kill format, PlySs2 (CF-301) synergized with a constant amount of DAP (2.5 μg/mL) over a range of sub-MIC concentrations (0.25 μg/mL to 0.025 μg/mL). Synergy is defined as a 2-log 10 (CFU/mL) or greater reduction at 24 hours. This represents a 64-fold reduction in the minimal sterilizing concentration of PlySs2 (CF-301) (as single agent) required for time-killing assays performed in human serum. Similarly, a strong synergy between PlySs2 (CF-301) and another antimicrobial agent, lysostaphin, in time-killing was observed. Here, each agent (0.005 mcg/mL) combination is sterile at the 24 hour time point. Remarkably, the minimum concentration of PlySs2 (CF-301) that synergizes with daptomycin in human serum (i.e., 0.025 μg/mL) is 160-fold lower than the minimum concentration of 4 μg/mL required for daptomycin synergy in CAMHB (Schuch R. et al.
al (2014) J Infect Dis 209(9):1469-1478), suggesting a potential link between host factors in human blood and the bactericidal activity of PlySs2 (CF-301).

A second method to confirm synergy was a checkerboard assay (Verma P (2007)
Methods for Determining Bactericidal Activity and Antimicrobial Interactions: Synergy Testing, Time-Kill Curves, and Population Analysis. Antimicrobial Susceptibility Testing Protocols, eds Schwalbe R, Steele-Moore L, & Goodwin AC (CRC Press, Boca Raton, FL), pp 275-298). A checkerboard was generated using a combination of PlySs2 (CF-301) below the MIC with either lysostaphin, daptomycin or vancomycin below the MIC against MRSA strain MW2 in either MHB or human serum. Synergy was defined as inhibitory activity greater than predicted by adding the two drugs together (ie, a minimum or average partial inhibitory concentration (FIC _MIN or FIC _AVG ) of 0.5 or less). PlySs2 (CF-301) showed very strong synergy with lysostaphin, daptomycin and vancomycin in human serum compared to MHB (Table 5). Considering the 32-fold difference in MIC values for PlySs2 (CF-301) in serum compared to MHB, the reduction in FIC values was even more pronounced. In extended analysis of eight additional MRSA strains, FIC _MIN and FIC _AVG values were consistently below 0.5 (ie, synergy) and superior to those determined in MHB (Table 6).

PlySs2 (CF-301) Shows Synergy with Human Lysozyme and Human Serum Albumin To understand the principles of human blood effects, a series of assays were used to test certain physical characteristics of the enhancer agent(s). Using a colorimetric-based MIC assay, we demonstrated that the enhancing effect of human serum was influenced by proteinase K (Fig. 7A) and was completely inactivated at temperatures above 75°C (Fig. 7B). Furthermore, the potentiation effect weakened at serum concentrations between 6.25% and 1.5% (Fig. 7C). These findings are consistent with the proteinaceous enhancer agent(s) being thermostable and abundant. Based on both these findings and the literature describing the potential of antibiotics and AMPs to synergize with host blood components, the antistaphylococcal activity of various potentially antibacterial blood proteins in combination with PlySs2 (CF-301) was next tested in a checkerboard assay using heat-inactivated human serum as the basal medium (Table 7). Although PlySs2 (CF-301) did not synergize with most of the agents tested (based on FIC _AVG values greater than 0.5), significant synergistic interactions were detected with both purified and recombinantly expressed forms of either human lysozyme (HuLYZ) or human serum albumin (HSA). FIC _AVG values below 0.1 were observed, consistent with very strong synergism in serum. Importantly, rabbit serum albumin (RabSA) synergized with PlySs2 (CF-301) (FIC _AVG <0.1), whereas rat serum albumin (RatSA) and mouse serum albumin (MSA) did not (FIC _AVG >1.16).

The effects of HuLYZ and HSA on PlySs2 (CF-301) activity were tested using an additional format. Initially, sub-MIC amounts of PlySs2 (CF-301) were combined with various HuLYZ concentrations from 1 μg/mL to 35 μg/mL using a time-killing assay (FIG. 8A). HuLYZ alone has no antistaphylococcal activity (21), but concentrations in human blood have been reported to vary from 1 μg/mL to 35 μg/mL (22). At HuLYZ concentrations above 5 μg/mL, a synergistic interaction was detected based on a 2-log 10 (CFU/mL) or greater reduction at 24 hours for the combination compared to PlySs2(CF-301) alone. In a second in vitro assay based on the decrease in optical density in treated cultures, addition of both HuLYZ (Fig. 8B) and HSA (Fig. 8C) over varying concentrations stimulated high levels of PlySs2 (CF-301) activity. Human lysozyme alone was completely inactive against Staphylococcus aureus at all concentrations tested. Similarly, HSA alone had no antibacterial activity in the absence of PlySs2 (CF-301). For HSA occurring in the blood at concentrations of approximately 40 mg/mL (7), maximal enhancement of PlySs2 (CF-301) activity was observed at HSA concentrations between 20 mg/mL and 40 mg/mL. A lytic assay is also the basis for determining the specific activity of PlySs2 (CF-301) (4). Activity of PlySs2 (CF-301) is typically observed at approximately 2500 units/mg protein, but addition of either HuLYZ or HSA results in a 9.8-fold or 17.8-fold increase in activity, respectively (Table 8). The fold increase in PlySs2 (CF-301) activity is 25% when HuLYZ and HSA are added together.

Various commercially available lysozyme and HSA reagents were used and tested with similar results as shown in Table 9 below.

Summary of Human Hematologic Effects with HuLYZ and HSA The criterion for human hematologic effects is a 32-fold reduction in MIC values determined in human serum compared to MHB. Using the MIC format, we sought to summarize human hematologic effects by adding either HuLYZ and/or HSA to two different media types lacking hematologic effects (i.e., MHB and MHB supplemented with 50% heat-inactivated human serum). The strongest lytic activity was observed with HuLYZ and HSA in combination with PlySs2 (CF-301). In MHB, addition of either HuLYZ (10 μg/mL concentration) or HSA (40 mg/mL concentration) resulted in 2-fold and 8-fold reductions in PlySs2(CF-301) MIC, respectively. Addition of both HuLYZ and HSA resulted in a 16-fold reduction (Table 10). The effect in MHB supplemented with 50% heat-inactivated human serum was similar (Table 11). Importantly, addition of RabSA produced an effect similar to that observed for HSA (ie, 8-fold reduction), whereas addition of either RatSA or MSA had little or no effect.

Interaction of PlySs2 (CF-301) with HSA in Human Serum Western blot analysis was performed using anti-PlySs2 (CF-301) antibody to detect PlySs2 (CF-301) in MIC wells of conditions with hematologic effect (i.e. human serum) and conditions without hematologic effect (i.e. MHB and MHB supplemented with 50% heat-inactivated human serum (MHB/HiHuS)). 1) was detected. In the presence of bacterial cells, PlySs2 (CF-301) forms a high molecular weight (approximately 150 kDA) band in human serum, but not in either MHB or MHB/HiHuS (Fig. 9A). The band at approximately 150 kDA differs from the commonly detected PlySs2 (CF-301) monomers and dimers (and trimers). Interestingly, a band at approximately 150 kDA is not observed when PlySs2 (CF-301) is incubated in human serum without bacterial cells (data not shown).

The effect of adding either HSA or RabSA (both 40 mg/mL) to MHB was tested. Addition of either HSA or RabSA partially established a hematologic effect in MHB (see above), resulting in the appearance of a band of approximately 150 kDa in the presence of bacterial cells (Fig. 9B). Addition of HuLYZ (10 μg/mL) is not associated with the formation of a band at ˜150 kDA (FIG. 9C).

The nature of the -150 kDa band formed in human serum was examined by mass spectrometry. In addition to other proteins commonly found at 150 kDa, the most abundant fragment signal detected was from human serum albumin (data not shown).

Serum lipids are required for the potentiating effect of PlySs2 (CF-301) Our observation that delipidated human serum does not synergize with PlySs2 (CF-301) (Table 3) suggests that fatty acids (FA) are required as part of the mechanism by which PlySs2 (CF-301) synergizes with HSA in serum. Most of the free FA in the circulation binds to HSA (24) and the delipidation process does not alter HSA levels, but reduces free FA levels by approximately a third (Sacks FM et al (2009) J Lipid Res 50(5):894-907). To confirm that low FA levels are responsible for the inability of PlySs2 (CF-301) to synergize in delipidated serum, the effect of introducing oleate and palmitate, two of the more common FAs in the circulation, on delipidated serum performance in the MIC assay format (Richieri GV & Kleinfeld AM (1995) J Lipid Res 36(2):229-240) was determined. Addition of either oleate or palmitate at a physiological concentration of 0.625 mg/mL resulted in an 8-fold and 16-fold reduction in PlySs2(CF-301) MIC, respectively, with no further reduction associated with simultaneous addition of both lipids (Table 12). Considering that FA alone (outside the serum context) had no effect on PlySs2 (CF-301) activity (data not shown), it is possible that this effect is mediated by HSA. This effect is also based on the understanding that FA bound to high affinity sites on HSA acts to promote additional avidity for drugs and other compounds (Yang F et al (2014) Int J Mol Sci 15(3):3580-3595).
It may also reflect a direct interaction with (CF-301).

Cell Surface Binding Studies with HuLYZ and PlySs2(CF-301) Confocal microscopy was used to test the binding of rhodamine-labeled PlySs2(CF-301) (CF-301 ^RHD ) to the surface of S. aureus strain ATCC 700699 pretreated with 40 mg/mL HSA (Figure 10). At a dose equivalent to 0.25-fold MIC, the CF-301 ^RHD construct showed extensive labeling of the staphylococcal cell wall in HSA-pretreated cells. No labeling was observed in the absence of HSA pretreatment. HSA pretreatment did not allow binding of the fluorophore-tagged lysins PlyG and PlyC (specific for the surface of B. anthracis and S. pyogenes, respectively) to Staphylococcus, confirming the specificity of HSA activity (data not shown).

The effect of pre-incubation of Staphylococci with different serotypes and MHB on subsequent labeling with 0.25-fold MIC amount of CF-301 ^RHD was also examined by fluorescence microscopy (Figure 11). Only pre-incubation with either human serum or rabbit serum resulted in extensive labeling of S. aureus strain MW2. Pre-incubation with rat serum, mouse serum or MHB alone resulted in poor labeling of cells observed only at longer exposure times. Addition of HSA to mouse serum restored high levels of binding and fluorescence.

Based on the ability of HuLYZ to synergize with PlySs2 (CF-301), confocal microscopy was then used to test the binding of Alexa Fluor-labeled HuLYZ (HuLYZ ^AF ) to Staphylococci pretreated with CF-301 in the sub-MIC range (FIG. 12). HuLYZ ^AF extensively labeled bacterial cell walls pretreated with either 0.5-fold or 0.25-fold MIC amounts of PlySs2 (CF-301). No labeling was observed in the absence of pretreatment with PlySs2 (CF-301). Furthermore, PlySs2 (CF-
301) Pretreatment did not promote binding of PIyG or PIyC lysins (data not shown).

Visualization of PlySs2 (CF-301) lytic activity in human serum Staphylococci were treated with various PlySs2 (CF-301) concentrations in either human serum or MHB for 10 minutes prior to analysis by transmission electron microscopy (TEM). Lytic activity was observed only at the highest concentration of PlySs2(CF-301) (ie, 5 μg/mL) in MHB, but widespread evidence of bacteriolysis was observed in all concentrations of human serum over a 100-fold range (FIG. 13). In addition to more rapid lysis in serum, lytic events were visually distinct compared to events in MHB. In human serum, all bacteria are encased in a proteinaceous sheath presumed to consist of host proteins, including HSA. Within the sheath, lytic bacteria are distinguished by peripheral lysis of electron-dense cell wall material, and interestingly, bacterial debris appears to remain sheathed. In contrast, the lytic event in MHB manifests as typical cell membrane bubbling and protrusion just prior to lysis.

In Vivo Evidence for Strong Hematologic Effects on PlySs2 (CF-301) Activity The efficacy of PlySs2 (CF-301) in addition to DAP was investigated using rat and rabbit models of infective endocarditis caused by MRSA strain MW2. In vivo studies showed that DAP plus PlySs2 (CF-301) was more effective in treating IE in a rabbit model compared to a rat model (Figure 14). In a rat model, a total dose of 10 mg/kg of PlySs2 (CF-301) administered in addition to a human therapeutic dose (HTD) equivalent of DAP produces an approximately 3 log10 (CFU/g) reduction in heart valve vegetation. The same 3 log10 reduction in bacterial density compared to DAP treatment alone is achieved in the rabbit model after administration of PlySs2 (CF-301) at a total dose of 0.09 mg/kg or greater plus less than the HTD equivalent of DAP. The difference in PlySs2(CF-301) efficacy in the two models is even more pronounced considering that a human equivalent dose of DAP was used in the rat model, while PlySs2(CF-301) was combined with a dose of DAP lower than the HTD equivalent in the rabbit model.

In the experimental rat IE model, an estimated AUC/MIC ratio of 0.87 or greater, achieved at a PlySs2 (CF-301) dose level of 10 mg/kg plus DAP, was required to achieve maximal efficacy (3 log10 (CFU/g) reduction in heart valve warts versus DAP alone) (Table 13 and Figure 15). Conversely, similar AUC/MIC values were obtained after a dose of 0.18 mg/kg PlySs2(CF-301) in a rabbit model. Overall, in vivo studies demonstrated that approximately 50-fold higher doses were required for similar bactericidal effects in rats (10 mg/kg) compared to rabbits (0.18 mg/kg).

A list of bacterial strains used in the studies herein is presented in Table 14 below.

Discussion In this study, the activity of PlySs2 (CF-301) and various other antibacterial lysins in Mueller-Hinton broth (the standard test medium) was compared to activities determined in biorelevant media types including human whole blood, plasma and serum.

Described and demonstrated herein is the substantial ability of PlySs2 (CF-301) to activate and synergize with a component of human blood and enhance the level of anti-staphylococcal activity relative to that predicted by AST according to standardized procedures (e.g., CLSI) using the CAMHB reference broth. The results of our study initially demonstrate a >100-fold reduction in the minimal sterilizing concentration of PlySs2 (CF-301) against 11 staphylococcal strains in blood matrices from up to 34 different sources compared to CAMHB in
It was based on an in vitro time-kill assay. We further demonstrated a greater than 32-fold reduction in MIC ₉₀ for 171 S. aureus isolates tested in human serum, and consistently low levels of MIC variability in blood, serum and plasma from 61 different human sources. Synergistic activity was observed in human serum at PlySs2 (CF-301) concentrations up to 160-fold lower than CAMHB in combination with either daptomycin or vancomycin in a time-kill and/or checkerboard format. Overall, these results support the potential efficacy of PlySs2 (CF-301) as an intravenously administered antimicrobial agent for the treatment of S. aureus bacteremia and endocarditis.

Furthermore, our findings suggest synergy between PlySs2 (CF-301) and two specific human blood components (i.e., lysozyme and albumin) that have no apparent (single-agent) endogenous antistaphylococcal activity as a major factor associated with enhanced activity and efficacy. The unique ability of PlySs2(CF-301) to activate and synergize with otherwise dormant bystanders (in terms of killing staphylococci) has important implications for the pharmaceutical use of PlySs2(CF-301) and the determination of its antimicrobial activity. Furthermore, our findings contrast sharply with the general understanding that for many systemically delivered conventional small-molecule antibiotics, protein binding in the circulation (primarily to albumin) acts to reduce drug activity (Zeitlinger MA, et al. (2011) Antimicrob Agents Chemother 55(7):3067-3074; Schmidt S, et al. (2008) Antimicrob Agents Chemother 55(7):3067-3074). 52(11):3994-4000, Burian A, et al. (2011)
J Antimicrob Chemother 66(1):134-137, Stratton CW & Weeks LS (1990) Diagn Microbiol Infect Dis 13(3):245-252, Beer J, Wagner CC, & Zeitlinger M (2009) AAPS J 11(1):1-12, Hegde SS, et al. (2004) Antimicrob Agents Chemother 4 8(8):3043-3050, Garonzik SM, et al. (2016) PLoS One 11(6):e0156131).

According to the studies and results herein, MIC data collected in serum may be more suitable for predicting in vivo activity for lysin polypeptides, particularly lysin polypeptides such as PlySs2 (CF-301) lysin, and for use in in vivo studies prior to clinical trials. In particular, based on this study, data collected in serum or with the addition of serum components may be more relevant for PlySs2 (CF-301), Sal lysins, lysostaphin, etc. presented herein with SH3-type binding domains, or other lysins, chimeras, constructs with SH3-type binding domains. In fact, the concentrations of certain peptides and peptide antibiotics (abx) required for in vivo therapy may be lower than conventionally estimated from MICs determined in artificial media. One improved approach for testing antimicrobial activity is the use of biorelevant concentrations of blood matrix.

The role of HSA in synergistically promoting the antimicrobial activity described for PlySs2 (CF-301) has not been previously described. Our hypothesis regarding HSA is strongly based on the following observations: (i) synergy with PlySs2 (CF-301) in multiple assay formats, including checkerboard, time-kill and lysis assays; (ii) ability to largely reconstitute serum effects by PlySs2 (CF-301) in media such as CAMHB/HiHuS and CAMHB; Putative interaction with PlySs2 (CF-301) detected by lot analysis; and (iv) enhancement of PlySs2 (CF-301) binding to the staphylococcal cell surface detected by microscopy. Additional support is obtained by using albumin from different species to replace HSA in replication experiments. In particular, rabbit SA can mimic the activity of HSA when used at a physiological concentration of 40 mg/mL. Rat SA and Mouse SA
can reproduce the HSA effect only at a supraphysiological concentration of 40 mg/mL. The relatively low physiological SA concentration in rodents of 20 mg/mL, which is half the normal physiological concentration of 40 mg/mL in rabbits and humans, may at least partially explain the inability of mouse and rat sera to act as a substrate for high levels of PlySs2 (CF-301) activity.

Our experiments with delipidated serum, particularly with the addition of oleate or palmitate, to reproduce the synergistic effect also indicate a direct interaction between PlySs2 (CF-301) and HSA as part of the mechanism for enhanced activity. Circulating HSA monomers are commonly complexed with lipids and have at least seven high- and intermediate-affinity FA-binding sites that can bind FAs and modulate and modify their interactions with antibiotics and other molecules (Yang F, Zhang Y, & Liang H (2014) Int J Mol Sci 15(3):3580-3595). PlySs2(
That such interaction with CF-301) occurs is also supported by our Western blot data showing the formation of SDS-resistant high molecular weight aggregates (i.e., ˜95 kDA and ˜150 kDA) only in the presence of HSA at conditions associated with 1 μg/mL PlySs2 (CF-301) MIC. PlySs2(CF-301) aggregates do not form in the absence of HSA at conditions associated with a PlySs2(CF-301) MIC of 32 μg/mL. The potential relationship between aggregate formation, HSA binding and high levels of PlySs2 (CF-301) activity contrasts with antibiotics whose activity is attenuated by binding to serum proteins.

S. aureus expresses Ebh, a large albumin-binding protein on its surface that contributes to survival in the blood and overall pathogenesis of staphylococcal infection (Cheng AG, Missiakas D, & Schneewind O (2014) J Bacteriol 196(5):971-981). Albumin-binding proteins are indeed found on a variety of pathogenic microorganisms that are theorized to adsorb HSA as part of their survival strategy in host tissues (Egesten A et al (2011) J Biol Chem 286(4):2469-2476). These findings are consistent with our observations, based on electron microscopy, showing rapid accumulation of a dense proteinaceous surface layer on S. aureus in human serum, which may consist of albumin and/or other blood components. This layer forms a visible sheath around Staphylococci that alters the visual manifestation of PlySs2 (CF-301)-mediated bacteriolysis (compared to events in CAMHB) and ultimately gives rise to bacterial ghost-like structures with often intact cell envelopes (Wu X, et al. (2017) Foodborne Pathog Dis 14(1):1-7), presumably encapsulated in a matrix of host-derived material. Encapsulation of bacterial debris and fragments (formed after lysis) may also play an important role in mitigating the potential risk of proinflammatory responses associated with the free release of these fragments into the host's bloodstream. Furthermore, encapsulation of bacteria and PlySs2 (CF-301) in human HSA matrices may reduce the risk of potentially adverse immune reactions. Overall, our findings support the hypothesis that the natural ability of staphylococci to coat themselves with HSA in the bloodstream, thereby evading human immunological surveillance, may be their only weakness with respect to HSA's ability to bind to PlySs2 (CF-301), leading to enhanced lysis. In other words, the synergistic mechanism of PlySs2(CF-301) and HSA is based on HSA-mediated improved accumulation kinetics for PlySs2(CF-301) on the bacterial cell surface, resulting in more rapid and efficient bacterial killing by PlySs2(CF-301).

The results presented here also allow conclusions regarding the ability of PlySs2 (CF-301) to activate lysozyme against Staphylococcus aureus in human serum. Initially, it is commonly understood that mature S. aureus peptidoglycan is resistant to HuLYZ activity by O-acetylation at the C-6 position of the cell wall N-acetylmuramic acid (Bera A et al (2005) Mol Microbiol 55(3):778-787, Bera A et al (2006) Infect Immun 74(8):
4598-4604). Therefore, no anti-staphylococcal activity was observed for HuLYZ tested alone over a wide range of concentrations in different AST formats. However, different synergistic antimicrobial activities of HuLYZ were observed when tested in combination with PlySs2 (CF-301) at physiological concentrations in time-kill, checkerboard and lysis assays. Although the contribution of HSA to the synergistic effect is greater based on in vitro replicated experiments, HuLYZ was consistently required for full replicated effect and was observed to dramatically increase the extent of PlySs2 (CF-301)-mediated surface labeling of Staphylococci by deconvolution microscopy. Combined with the proposed activity of HSA, our model suggests that PlySs2 (CF-301) selectively accumulates on the cell surface upon interaction with HSA and independently upon HuLYZ activation. The exact nature of this HuLYZ activity is unknown, but one possible explanation is that PlySs2 (CF-301)-mediated cleavage of peptidoglycan initiates access of HuLYZ to the nascent peptidoglycan formed prior to O-acetylation, and subsequent hydrolytic activity of HuLYZ promotes more PlySs2 (CF-301) binding as lysis progresses.

To understand the in vivo efficacy profile of PlySs2(CF-301) in the targeted clinical indications of staphylococcal bacteremia and endocarditis, experiments will be conducted in two species (rabbit and rat) where differences in the ability of PlySs2(CF-301) to synergize with their respective serotypes in the ex vivo format reported here are observed. The IE model has been established in both rabbits and rats (Abdelhady W et al (2017) Antimicrob Agents Chemother 61(2), Hady WA, Bayer AS, & Xiong
YQ (2012) J Vis Exp (64):e3863), has a prominent biofilm component that is highly relevant for the clinical indications of interest for PlySs2 (CF-301). Based on our ex vivo observations of potent PlySs2 (CF-301) synergy in rabbit serum, but not in rat serum, we observed that more than 50-fold higher PlySs2 (CF-301) doses and more than 20-fold higher AUC exposures were required to obtain similar bactericidal effects in rats (10 mg/kg) compared to rabbits (0.18 mg/kg). These models provide further evidence for the potential therapeutic significance of PlySs2(CF-301)'s ability to activate and synergize with HSA and HuLYZ and support the predicted efficacy of PlySs2(CF-301) at doses selected for therapeutic use in clinical trials evaluating PlySs2(CF-301) for the treatment of S. aureus bacteremia, including endocarditis.

Several previous studies have evaluated antimicrobial efficacy in serum and plasma, but with mixed results and conclusions. The presence of human plasma has been reported to increase the activity of antimicrobial peptidomimetics (AMPs), such as α-peptide/β-peptoid peptidomimetics against E. coli (Hein Kristiansen et al 2013, Citterio et al 2016), leading to the hypothesis that synergy with blood components may be involved, but this has not been fully evaluated. The findings that peptidomimetics and PMB lowered MICs in plasma by 2- to 16-fold lead to the suggestion that potentiation by plasma may be caused by endogenous blood components such as complement, as heat inactivation abolished synergism. Heat inactivation caused a dramatic increase in MIC. It has been suggested that other components of plasma, including proteins of the complement cascade, may act in potentiation, which may explain why plasma produces more potentiation with some AMPs than serum. Unlike serum, plasma contains active clotting factors that can respond to the presence of bacteria.

Plasma enhanced the activity of PMB, but not gentamicin and ampicillin. Other studies have reported that complement proteins can act synergistically with antimicrobial compounds such as PMB and AMP. The potentiating effect depends on the mode of action of the agent, since only compounds that are active on the cell membrane or coat appear to be potentiated. It was concluded that coagulation proteins interacted with complement to enhance its activity.

Vaara et al 1984 evaluated the outer-membrane-disrupting peptide PBMN and found that the small cationic outer-membrane-disrupting peptide PMBN sensitized E. coli to the bactericidal action of serum and facilitated the insertion or binding of antibodies or other factors present in normal serum through activation of the resulting complement cascade. PMBN-mediated bactericidal activity of serum was abolished by heating. This effect was observed in humans, guinea pigs and rabbits, moderate effect in rats and no effect in mouse serum. Mice are cited as unique among mammals because neither normal or hyperimmune sera nor peritoneal fluids lack bactericidal action and are easily killed in sera of other animals.

Pruul and McDonald 1992 assessed potentiation of azithromycin's antibacterial activity by normal human serum. In the Pruul and McDonald study, 40% of sera caused a 15-fold reduction in MIC against the bacterium Staphylococcus aureus in the MIC assay. However, this enhancement was not inhibited by heat inactivation, indicating that it is not heat sensitive. Also, Pruul and McDonald found no difference between complement-inactivated serum or antibody-depleted serum. Also, addition of albumin (Cohn Fraction V) to the TSB test broth (up to 70 mg/ml) did not restore activity. This effect was restricted to Gram-negative species and was not observed in Staphylococci.

Serum-enhancing factors of human serum can include serum lipoproteins, pH changes, antimicrobial peptides. Changes in bacterial growth rate and bacterial surface composition induced by serum factors can alter bacterial membrane permeability, alter antibiotic accumulation kinetics, or enhance antibiotic binding.

Given the considerable diversity of innate immune molecules in animal hosts, it is possible that PlySs2 (CF-301) activity synergizes with many other peptides to improve their overall efficacy.

Based on this research, we propose the following model to explain the enhanced activity biologically. In blood, S. aureus bacteria are coated with serum proteins, including HSA, to evade the immune system (HSA-binding proteins on S. aureus are known and previously described). HSA binding comes at a considerable cost, including reduced cross-linking. The interaction of PlySs2 (CF-301) (and other SH3-binding lysins) with HSA in the presence of bacteria concentrates the lysins on the bacterial surface. This concentration, combined with the reduction in cross-linking exhibited by HSA binding, enhances the antimicrobial activity of PlySs2 (CF-301). In the case of human lysozyme (HuLYZ), which is normally ineffective against Staphylococcus aureus bacteria, enhanced activity of PlySs2 (CF-301), combined with reduced cross-linking, promotes HuLYZ binding and activity against nascent peptidoglycans that remain susceptible to HuLYZ. The combined activity of HSA, HuLYZ and PlySs2 (CF-301) enables blood effects.

Sensitization of S. aureus to HuLYZ is newly recognized and has not been previously observed. A hallmark of S. aureus is resistance to lysozyme, which is central to its immune evasion strategy. PlySs2(CF-301) treatment circumvents this and renders lysozyme active against S. aureus.

Thus, this experiment demonstrates that PlySs2 (CF-301) is a highly efficient anti-staphylococcal agent based on its intrinsic hydrolytic activity and its ability to enhance the antibacterial activity of lysozyme. In addition, PlySs2 (CF-301) synergizes with and potentiates HSA and/or uses HSA binding to bacterial cells to promote PlySs2 (CF-301).
(CF-301) to the bacteria and/or effectively localize PlySs2 (CF-301) to the site of bacterial infection.

Our data demonstrate that the human blood milieu synergizes with the antistaphylococcal lysin PlySs2 (CF-301) and other exemplary SH3-type binding domain-containing lysins, greatly enhancing or potentiating their antimicrobial activity, as a result of synergistic interactions with both the innate immune effector lysozyme and the most abundant serum protein albumin. This study highlights the striking suitability of this enzyme class for handling widely different peptidoglycan substrates. This observation highlights the fitness of microorganisms in response to antibiotic challenge and demonstrates an established antibiotic susceptibility that has long been thought to rule out resistance.

Materials and Methods Bacteria, media and growth conditions. A subset of bacterial strains used in this study are listed in Table S8. The 171 S. aureus strains used in Table 1, including 74 MSSA and 75 MRSA clinical isolates from 2011 obtained from JMI Laboratories (North Libery, IA), have been previously described (4). Bacteria were grown on BBL™ Trypticase™ Soy Agar (TSAB; Becton, Dickinson & Company (BD)) containing 5% sheep blood, cation-adjusted BBL™ Mueller-Hinton II Broth (CAM), unless otherwise specified.
HB; BD), or brain heart infusion broth (BHI; BD). The sources, descriptions and lot numbers of all human and animal blood matrices tested, with the exception of HyClone™ Fetal Bovine Serum (0.1 micron filtered; GE Healthcare Lifesciences), are listed in Tables S1 and S2. Staphylococci were grown at 37° C. with aeration unless otherwise specified.

reagent. Lysin PlySs2 (CF-301) was expressed, purified and stored as previously described (4). Human anti-PlySs2 (CF-301) IgG3 monoclonal antibody (expressed and purified at ContraFect Corporation) and mouse anti-human IgG3 heavy chain antibody, AP conjugate (05-3622 from ThermoFisher) were used at 1:1000 dilution as primary and secondary antibodies for Western blots. All agents (and vendor sources) tested in combination with PlySs2 (CF-301) are as follows: β-defensin-3, human (Anaspec); Leap-1, human (Anaspec); Leap-2, human (GenScript); LL-37, human (Anaspec); ); HNP-1, human (Anaspec);
HNP-2, human (Anaspec); human platelet factor IV 18 (Anaspec); lactoferrin, human milk (Sigma-Aldrich); lactoferrin, bovine colostrum (Sigma-Aldrich); lactoferricin H, human (Anaspec); human lysozyme, recombinant expression in rice (Sigma-Aldrich); human serum albumin, fraction V (Sigma-Aldrich); human serum albumin, fraction V, fatty acid-free, globulin-free (Sigma-Aldrich); human serum albumin, recombinantly expressed in yeast (Albumin Biosciences); mouse serum albumin, recombinantly expressed in yeast (Albumin Biosciences); Sigma-Aldrich); and rat serum albumin (Sigma-Aldrich). Sodium oleate, sodium palmitate, vancomycin hydrochloride, daptomycin, proteinase K-agarose from Tritirachium album were obtained from Sigma-Aldrich. For microscopy, DAPI, Alexa Fluor™ 488 and NHS-rhodamine were obtained from Thermo Fisher Scientific. Labeling and purification of PlySs2 (CF-301) conjugated to NHS-rhodamine and HuLYZ conjugated to Alexa Fluor 488 were performed as described in the manufacturer's protocol. Unreacted fluorophore was removed using PD-10 desalting columns (GE Healthcare) and labeling efficiency was determined to be >80% in all cases. The activity of PlySs2(CF-301)-rhodamine (CF-301 ^RHOD ) was confirmed to be comparable to PlySs2(CF-301) using standard MIC assays. The activity of HuLYZ-Alexa Fluor 488 (HuLYZ ^AF ) was confirmed to be comparable to HuLYZ using a drop dilution assay on 1% agarose surfaces (Sigma-Aldrich) impregnated with 1 mg/ml peptidoglycan from Micrococcus luteus. The production, purification and use of GFP-tagged PlyG (PlyG ^GFP ) and Alexa Fluor488-tagged PlyC (PlyC ^AF ) have been previously described (38, 39).

Time-killing assay. Bactericidal activity was tested according to the method of CLSI (52). Assays were performed with a bacterial inoculum of 5×10 ⁵ CFU/mL in CAMHB or the indicated (undiluted) blood matrix in 125 mL glass Erlenmeyer flasks with agitation. The indicated agents were tested over a 10-fold range of concentrations. For daptomycin, CAMHB cultures were supplemented with 50 μg/mL Ca ²⁺ . A growth control with buffer alone was always included. Immediately prior to treatment and at designated time intervals up to 24 hours thereafter, culture aliquots were removed and diluted with activated charcoal (to prevent or stop drug activity). Ten-fold serial dilutions of inactivated cultures were then plated on TSAB and incubated at 37° C. for 24 hours prior to colony counting. Bactericidal activity was defined as a reduction of 3 log10 (CFU/mL) or greater relative to the initial inoculum.

MIC assay. MIC values were determined by broth microdilution using CLSI's reference method (62) in either CAMHB or the indicated blood matrices. CAMHB/HiHuS was prepared by adding 50% human serum to CAMHB, followed by filtration through a Microcon Centrifugal Filter Unit (Amicon Ultra-15; Millipore) with a 50 kDa cutoff, followed by 7 minutes to completely inactivate the component(s) involved in synergy with PlySs2 (CF-301). Prepared by incubating at 0° C. for 20 minutes. For addition of fatty acids to delipidated serum, 50 mg/mL stock solutions of either oleate in H ₂ O or palmitate in 100% ethanol were prepared and added to serum to achieve a final concentration of 0.625 mg/mL. The spiked serum was then incubated at 37° C. for 1 hour before being used to promote fatty acid binding to HSA. Colorimetric determination of PlySs2 (CF-301) MIC values was performed using AlamarBlue™ (Thermo Fisher Scientific) according to the manufacturer's protocol exactly. Analysis of PlySs2 (CF-301) activity in human serum pretreated with proteinase K-agarose beads for 3 hours was performed by the manufacturer (Sigma-Aldrich
) was performed according to the protocol of As a control for protease carryover after removal of proteinase K-agarose beads, treated serum was diluted 3:4 with untreated serum prior to MIC determination. Addition of untreated serum was expected to restore synergy only in the absence of carryover of either unbound proteinase K or proteinase K-agarose beads.

Lysis assay. An overnight culture of MRSA strain MW2 was diluted 1:100 in BHI and grown for 2.5 hours at 37° C. with aeration. Log-phase cells were washed, concentrated 10-fold in 20 mM phosphate buffer (pH 7.4), and divided into equal aliquots to which varying concentrations of either HSA (Albumin Biosciences) or lysozyme from human neutrophils (AVIVA Bioscience) were added. For each concentration of HSA or lysozyme, 0.1 mL of the mixture was aliquoted in duplicate into 96-well flat bottom non-tissue culture treated microtiter plates (BD). The lysis reaction was then initiated by adding 0.1 mL of PlySs2 (CF-301) (in phosphate) to all wells to a final concentration of 4 μg/mL. Pl in control wells
ySs2(CF-301), HSA or HuLYZ alone were added at appropriate concentrations. Samples were mixed and optical density at 600 nm ( _OD600 ) was followed for 15 minutes at room temperature in a SpectraMax M5 microplate reader (Molecular Devices).

A modified lytic assay was performed to determine PlySs2 (CF-301) specific activity. Log-phase MW2 cells were prepared as described above and split into 4 mL aliquots containing either phosphate buffer (reactions with cells alone, no agent added) or HSA (Albumin Biosciences) and/or human lysozyme (AVIVA Biosciences). PlySs2 (CF-301
) (starting at 0.5 mg/mL) was serially diluted two-fold with 20 mM phosphate (pH 7.4) in 0.1 mL volumes across rows 1-11 of a 96-well plate. Column 12 contained no enzyme, only buffer, and was used as an assay control well. Bacterial cells mixed with either HuLYZ buffer, HSA, lysozyme, or HSA were added as 0.1 mL aliquots to each well of three rows of serially diluted PlySs2 (CF-301). Each plate was read at 600 nm at room temperature for 15 minutes (3 seconds shaking between readings) using a microplate reader as described above. The specific activity (units/mg) of PlySs2(CF-301) was determined based on the PlySs2(CF-301) dilution showing curves just above and below the optical density of 50% of the buffer control at 15 minutes.

Checkerboard assay. The checkerboard assay was performed as described (66) and adapted by CLSI's method for broth microdilution (62). A checkerboard was constructed by first dispensing equal volumes of two-fold diluted PlySs2 (CF-301) into each row of a 96-well polystyrene microtiter plate along the x-axis. A separate plate was created with corresponding rows along the y-axis in which each well contained the same amount of a two-fold dilution of another agent. The dilutions were then combined such that each column contained a fixed amount of PlySs2(CF-301) and a two-fold dilution of the second agent and each row contained a fixed amount of the second agent and a two-fold dilution of PlySs2(CF-301). Each well thereby contains a unique combination of PlySs2 (CF-301) and the second agent. Bacteria were isolated approximately 5× in CAMHB or human serum (pooled male, type AB, sterile filtered, US origin; Sigma-Aldrich).
A concentration of 10 ⁵ CFU/mL was added to each well. The MIC of each drug alone and in combination was recorded after 18 hours at 37°C in ambient air. Results are expressed in units of ΣFIC index, which is equal to the sum of FIC for each drug. The FIC of a drug is defined as the MIC of the combined drug divided by the MIC of the drug used alone. Both ΣFIC _min (lowest ΣFIC value obtained for all combinations) and ΣFIC _AVG (average ΣFIC value for three consecutive drug combinations) are reported for each agent pair. Combinations are interpreted as synergistic when the ΣFIC index is less than or equal to 0.5, additive when greater than 0.5 and less than or equal to 2, and antagonistic when greater than 2 (16). Colorimetric determination of PlySs2 (CF-301) MIC values was also performed using AlamarBlue™ (Thermo Fisher Scientific) according to the manufacturer's protocol.

Synergy time-killing assay. Synergy time-killing curves were performed according to the method described by CLSI (52). Strain MW2 was suspended in CAMHB containing 50% HiHuS at a concentration of 5×10 ⁵ colony forming units (CFU)/mL and exposed to PolySs2 (CF-301) and/or daptomycin, HSA (Albumin Biosciences) and HuLYZ (AVIVA Bioscience) for 24 h at 35° C. with ambient agitation. At designated time intervals, culture samples were removed, serially diluted and plated to determine CFU/mL. The resulting kill kinetic determinations are graphically represented by plotting log ₁₀ (CFU/mL) against time. Synergy was defined as a 2-log ₁₀ (CFU/mL) or greater reduction between the combination and its most active component, the lowest component tested at an ineffective concentration.

Fluorescence Microscopy: Binding of PlySs2 (CF-301) to Bacterial Cell Surfaces in Different Serum Environments. Mid-logarithmic MRSA strain MW2 was suspended at 1×10 ⁷ CFU/mL in either CAMHB or 100% serum from either human (Sigma-Aldrich), rabbit (Gibco), rat (BioreclamationIVT) or mouse (BioreclamationIVT) sources,
Incubated for 30 minutes at 37°C. 40 mg/mL HSA (Albumin Biosciences
) were also tested. After pre-incubation, bacteria were washed with 1× phosphate-buffered saline (PBS), resuspended in 50 μl of PBS, and allowed to adhere to the surface of poly-L-lysine-coated coverslips. Cells were washed and treated with CF-301 ^RHOD (2 μg/mL in PBS) for 10 min, then washed and counterstained with DAPI. Slides were mounted in 50% glycerol and 0.1% p-phenylenediamine in PBS (pH 8). Fluorescence microscopy was performed using a Nikon Eclipse E400 microscope equipped with a Nikon 100×/1.25 oil immersion lens and a Retiga EXi fast 1394 camera (QImaging). QCapture Pro version 5.1.1.14 software (QImaging) was used for image capture and processing.

Fluorescence microscopy: enhancement of PlySs2 (CF-301) binding in the presence of HSA. Overnight cultures of VISA strain ATCC 700699 were diluted 1:100 in CAMHB, grown to an OD ₆₀₀ of 0.6 and attached to the surface of poly-L-lysine coated coverslips. Cells were washed with PBS and treated with 10 μl of PBS containing HSA (Albumin Biosciences) or PBS alone for 30 minutes at room temperature. Duplicate samples were then added with 1/10 the original volume of PBS or PBS containing NHS-rhodamine labeled PlySs2 (CF-301) to a final concentration of 4 μg/mL (0.25×MIC). Cells were incubated for an additional 30 minutes at room temperature, washed with PBS, and fixed with 2.6% paraformaldehyde in PBS for 45 minutes at room temperature. Slides were then washed with PBS and mounted in PBS (pH 8.0) containing 50% glycerol and 0.1% p-phenylenediamine. DAPI was used as a counterstain dye in all assay conditions. Deconvolution microscopy was performed using a DeltaVision image reconstruction microscope (Applied Precision/Olympus Corporation) equipped with a CoolSnap QE cooled CCD camera (Photometrics). Imaging was performed on an Olympus Corporation 100x/1.40 N.V. A. , was performed with a UPLS Apo oil immersion objective. Z-stacks were acquired at 0.15 μm intervals. Images were deconvolved using SoftWoRx software (Applied Precision/DeltaVision)
Corrected for chromatic aberration and presented as a maximum intensity projection combining all relevant z-sections. In complementary assays similar to those above, NHS-rhodamine labeled PlySs2 (CF-301) was replaced with either 25 μg/mL PlyG ^GFP or PlyC ^AF . After processing, samples were visualized by fluorescence microscopy using a Nikon Eclipse E400 microscope equipped with a Nikon 100×/1.25 oil immersion lens.

Fluorescence microscopy: enhancement of HuLYZ binding in the presence of PlySs2 (CF-301). Overnight cultures of VISA strain ATCC 700699 were diluted 1:100 in CAMHB, grown to an OD ₆₀₀ of 0.6 and attached to the surface of poly-L-lysine coated coverslips. Cells were washed with PBS and treated with 50 μl of PBS containing different concentrations of PlySs2 (CF-301) or PBS alone for 30 min at room temperature. Duplicate samples were then added with 1/10 the original volume of PBS or PBS containing Alexa Fluor 488 labeled HuLYZ to a final concentration of 10 μg/mL. Cells were incubated for an additional 30 minutes at room temperature, washed with PBS, and fixed with 2.6% paraformaldehyde in PBS for 45 minutes at room temperature. Slides were then washed with PBS and mounted in 20 mM Tris (pH 8.0), 90% glycerol, 0.5% n-propyl gallate. DAPI was used as a counterstain dye in all assay conditions. Deconvolution microscopy was performed as described above using a DeltaVision image reconstruction microscope (Applied Precision/Olympus Corporation) equipped with a CoolSnap QE cooled CCD camera (Photometrics). In complementary analyses, Alexa Fluor 488-labeled CF-301 was replaced with either 25 μg/mL PIyG ^GFP or PIyC ^AF . After processing, samples were visualized by fluorescence microscopy using a Nikon Eclipse E400 microscope equipped with a Nikon 100×/1.25 oil immersion lens.

electron microscopy. Mid-logarithmic strain M growing in CAMHB or human serum (Sigma-Aldrich)
W2 were treated with indicated concentrations of PlySs2 (CF-301) or buffer alone (control) for 15 minutes at 37°C. Cells were then washed with 1× phosphate buffer (PB) and resuspended in a solution of 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Samples were post-fixed in 1% osmium tetroxide, block stained with uranyl acetate, and processed according to standard procedures by Rockefeller University Electron Microscopy Service. Tecnai™ Spirit BT
Samples were visualized using transmission electron microscopy (FEI). Human serum was sterile filtered and AB
It was obtained from a pooled male population of blood type US origin (approximately 70 subjects).

Western blot analysis. PlySs2 (CF-301) MIC well samples taken from the indicated media types were analyzed by Western blot. Each 10 μl sample aliquot was run on a 4%-12% Tris-glycine minigel (Novex™) and then transferred to polyvinylidene fluoride (PVDF) membrane by electroblotting. The PVDF membrane was incubated with an anti-PlySs2 (CF-301)-specific protein G affinity purified human IgG3 recombinant monoclonal antibody followed by a secondary monoclonal mouse anti-IgG3-alkaline phosphatase conjugated detection antibody. Both primary and secondary antibodies were used at 1:1000 dilution. Membranes were stained with nitroblue tetrazolium and 5-bromo-4-chloro-3'-indolylphosphate (NBT/BCIP) chromogenic substrates. Molecular weights of visible bands were determined by comparison with bands of molecular weight standards run in the same gel.

Rat infectious endocarditis model. Sprague-Dawley rats (250-275 g) anesthetized by intraperitoneal injection (IP) of a cocktail of 87 mg/kg ketamine and 13 mg/kg xylazine underwent standard placement of a transcarotid transaortic valve catheterization into the left ventricle. 48 hours later, animals were challenged with S. aureus strain MW2 (approximately 1 x ¹⁰⁵ colony forming units (CFU)/rat; IV) to induce endocarditis. Twenty-four hours after infection, a cohort of rats was euthanized, heart valve warts were harvested and initial tissue burden was determined (control group). The remaining rats were treated with either vehicle (saline; single IV dose), or DAP alone (40 mg/kg; subcutaneous injection (SQ); qd x 4 days), or DAP plus PlySs2 (CF-301). PlySs2 (CF-301) was administered IV as a single slow bolus (injection over 5-10 minutes) at four dosing schedules (1 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg) immediately after the initial DAP dose only on the first day of treatment. Treated animals were euthanized (200 mg/kg pentobarbital sodium by rapid IP push) 24 hours after the last DAP treatment (5 days post-infection), heart valve warts were excised, weighed, homogenized and serially diluted in sterile PBS for quantitative culture on TSAB. All culture plates were incubated at 37° C. for 24 hours and the resulting colonies were counted and expressed as log ₁₀ (CFU/g tissue). Data for each organ in different treatment groups were calculated as the median of log ₁₀ (CFU/g of tissue) ±95% CI.

Population PK modeling of PlySs2 (CF-301). Pharmacokinetic (PK) data were collected from previous non-clinical toxicology studies involving rats and dogs treated with various doses of PlySs2 (CF-301) (41,42). Post-dose plasma was collected over time and analyzed using a validated PlySs2 (CF-301) ELISA. Predicted AUC values for PlySs2(CF-301) at doses of 1 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg as a 10-minute intravenous infusion were derived from population PK modeling based on non-clinical rat and dog PK experiments. For the present study, the previously reported AUC values were divided by the MIC value determined in rat serum for MRSA isolate MW2 (i.e., 16 μg/mL) to obtain the calculated AUC/MIC ratio values reported in Table 13.

Rabbit infectious endocarditis model. New Zealand White rabbits (2.2-2.5 kg) anesthetized by intramuscular injection (IM) of a cocktail of 35 mg/kg ketamine and 5 mg/kg xylazine underwent standard placement of a transcarotid-transaortic valve catheterization into the left ventricle (43). Forty-eight hours after catheterization, animals were IV challenged with an inoculum of approximately 2×10 ⁵ CFU of S. aureus strain MW2 to induce infective endocarditis (IE). Previous studies have shown that this inoculum induces IE in >95% of catheterized animals. Twenty-four hours after infection, a cohort of rabbits was euthanized, heart valve warts were harvested and initial tissue burden was determined (control group). The remaining rabbits were treated with either vehicle (saline; single IV dose), or DAP alone (4 mg/kg IV; qd x 4 days), PlySs2 (CF-301) alone, or DAP plus PlySs2 (CF-301). PlySs2 (CF-301) was administered IV as a single slow bolus (injection over 5-10 minutes) at 4 dosing schedules (0.09 mg/kg, 0.18 mg/kg, 0.35 mg/kg, 0.70 mg/kg and 1.4 mg/kg) immediately following the initial DAP dose only on the first day of treatment. Treated animals were euthanized (200 mg/kg pentobarbital sodium by rapid IP push) 24 hours after the last DAP treatment (5 days post-infection) and heart valve warts were removed, weighed, homogenized and serially diluted in sterile PBS for quantitative culture on TSAB. All culture plates were incubated at 37° C. for 24 hours and the resulting colonies were counted and expressed as log ₁₀ (CFU/g tissue). Data for each organ in different treatment groups were calculated as the median of log ₁₀ (CFU/g of tissue) ±95% CI.

Rabbit pharmacokinetics. New Zealand White rabbits were dosed with PlySs2 (CF-301) at 0.18 mg/kg, 0.35 mg/kg, 0.07 mg/kg or 1.4 mg/kg (IV, slow bolus). Plasma was collected after titrating hours of dosing and analyzed using a validated PlySs2 (CF-301) ELISA as described (63, 64). Predicted AUC values for PlySs2(CF-301) at doses of 1 mg/kg, 2.5 mg/kg, 5 mg/kg and 10 mg/kg as a 10 minute intravenous infusion were derived using WinNonLin (data not shown). These values were divided by the MIC value for MRSA isolate MW2 (1 μg/mL in rabbit serum) to calculate the AUC/MIC ratio values reported in Table 8.

The theory of daptomycin dosing in rabbits. Daptomycin dose-response experiments were performed once daily for 4 days at doses ranging from 1 mg/kg to 10 mg/kg (IV) in the rabbit IE model caused by S. aureus strain MW2. Daptomycin at 4 mg/kg, representing a sub-therapeutic dose in humans, was chosen to explore the benefits of PlySs2 (CF-301) therapy in addition to daptomycin. In the rabbit IE model, a 4 mg/kg (IV) daptomycin dose produced approximately a 2-3 log ₁₀ reduction in bacterial load compared to vehicle-treated controls. Treated animals still had a significant load of approximately 5-7 log ₁₀ , resulting in a significant dynamic range and an additive effect of PlySs2 (CF-301) on this treatment regimen was observed.

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Example 2
It is noteworthy that the lysin polypeptides PlySs2 (CF-301), lysostaphin (LSP) and Sal lysin, which exhibit serum component effects specifically via serum albumin and lysozyme, are similar in that they all have SH3-type binding domains. To further evaluate the binding domains associated with serum component effects, various chimeras were constructed by substituting or exchanging one binding domain for another. Chimeric lysins having a Sal1 CHAP catalytic domain with a ClyS binding domain, a PlySs2 (CF-301) binding domain or a lysostaphin binding domain were assayed for MIC using MHB against human serum. The results are shown in Table 14 below. The binding domain of ClyS does not support blood effects, whereas the binding domain of PlySs2 (CF-301), an SH3-type binding domain, supports blood effects. Similarly, the SH3-type binding domain of lysostaphin also exhibits serum component effects and retains serum effects when fused to the Sal1 catalytic domain as a chimeric lysin.

This invention may be embodied in other forms or carried out in other ways without departing from its spirit or essential characteristics. Accordingly, the present disclosure is to be considered illustrative and not restrictive in all its aspects, and all changes that come within the meaning and range of equivalents of the invention, as indicated by the appended claims, are intended to be embraced therein.

Various references are cited throughout this specification, each of which is hereby incorporated by reference in its entirety.

Claims (39)

A composition or combination for the enhanced or synergistic killing of Gram-positive bacteria comprising an isolated lysin polypeptide having an SH3-type binding domain and one or more blood component proteins selected from serum albumin and lysozyme, or peptides or fragments thereof, wherein the peptides or fragments exhibit the activity of a full-length albumin or lysozyme protein with respect to the lysin polypeptide activity-enhancing effect. 2. The composition or combination of claim 1, wherein said lysin polypeptide having an SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. 2. The composition or combination of claim 1, wherein the lysin polypeptide having a SH3-type binding domain is PlySs2 (CF-301) lysin, comprising the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill Staphylococcus and Streptococcus bacteria. 2. The composition or combination of claim 1, wherein said serum albumin is selected from human serum albumin, horse serum albumin, dog serum albumin, rabbit serum albumin, rat serum albumin or calf (bovine/bovine subfamily) serum albumin. 2. The composition or combination of claim 1, wherein said lysozyme is human lysozyme. 2. The composition or combination of claim 1, wherein said blood components serum albumin and/or lysozyme have no or limited endogenous antibacterial, in particular antistaphylococcal, activity in the absence of said lysin polypeptide. 3. The composition or combination of claim 1, further comprising one or more serum fatty acids. 8. A composition or combination according to claim 7, wherein said serum fatty acids are selected from oleate and palmitate. 1. A method of killing or reducing a population of Gram-positive bacteria comprising contacting said bacteria with a composition comprising an isolated lysin polypeptide having an SH3-type binding domain and one or more blood components selected from serum albumin and lysozyme in an amount effective to kill said Gram-positive bacteria. 10. The method of claim 9, wherein said serum albumin is selected from human serum albumin, horse serum albumin, dog serum albumin, rabbit serum albumin, rat serum albumin and calf (bovine/bovine subfamily) serum albumin. 10. The method of claim 9, wherein said serum albumin is human serum albumin or a fragment or peptide thereof capable of binding Gram-positive bacteria. 10. The method of claim 9, wherein said lysozyme is human lysozyme. 10. The method of claim 9, wherein said Gram-positive bacteria are Staphylococci or Streptococci. 10. The method of claim 9, wherein said Gram-positive bacterium is Staphylococcus aureus. 10. The method of claim 9, wherein the lysin polypeptide having an SH3-type binding domain is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, Phil lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. 10. The method of claim 9, wherein the lysin polypeptide having a SH3-type binding domain is PlySs2 (CF-301) lysin, comprising the amino acid sequence set forth in SEQ ID NO:3 or a variant thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill Staphylococcus and Streptococcus bacteria. 10. The method of claim 9, wherein said bacteria are further contacted with serum fatty acids optionally selected from oleate and palmitate. 9. A method of treating an antibiotic-resistant Staphylococcus aureus infection in a human comprising administering to a human having an antibiotic-resistant Staphylococcus aureus infection an effective amount of a composition or combination according to any one of claims 1-8. 1. A method of treating an antibiotic-resistant Staphylococcus aureus infection in a human comprising administering to a human with an antibiotic-resistant Staphylococcus aureus infection an effective amount of a composition comprising an amount of an isolated lysin polypeptide having an SH3-type binding domain and capable of killing Gram-positive bacteria, and human lysozyme. 20. The method of claim 19, further comprising assessing the level of human serum albumin at the site of infection and/or assessing coverage of said antibiotic-resistant Staphylococcus aureus by serum albumin at the site of infection, and further comprising administering human serum albumin, or a fragment or peptide thereof capable of binding Gram-positive bacteria, wherein said lysin polypeptide and lysozyme are effective in killing said antibiotic-resistant Staphylococcus aureus. A chimeric or fusion polypeptide comprising a lysin-derived SH3-type bacterial binding domain and human serum albumin or a fragment or peptide thereof capable of binding Gram-positive bacteria. 22. The chimeric or fusion polypeptide of claim 21, further comprising the lytic domain of human lysozyme. A chimeric or fusion polypeptide comprising a lysin-derived SH3-type bacterial binding domain and human lysozyme or a fragment or peptide thereof capable of lysing Gram-positive bacteria. 24. The chimeric or fusion polypeptide of claim 23, further comprising human serum albumin, or a fragment or peptide thereof capable of binding Gram-positive bacteria. A method of enhancing the antimicrobial activity of an antimicrobial agent or peptide, comprising administering the peptide in combination with a lysin polypeptide having an SH3-type binding domain and human serum albumin, or a fragment or peptide thereof capable of binding Gram-positive bacteria. 26. The method of claim 25, further comprising administering human lysozyme, or a fragment or peptide thereof capable of lysing Gram-positive bacteria. A method of enhancing the antimicrobial activity of an antimicrobial agent or peptide, comprising administering the peptide in combination with a lysin polypeptide having an SH3-type binding domain, and lysozyme, or a fragment or peptide thereof capable of lysing Gram-positive bacteria. 29. The method of claim 28, further comprising administering human serum albumin, or a fragment or peptide thereof capable of binding Gram-positive bacteria. A method of susceptibility testing of Gram-positive bacteria comprising evaluating an antimicrobial peptide in broth, assay medium or solution supplemented with serum albumin. 30. The method of claim 29, wherein the broth, assay medium or solution is supplemented with human serum albumin, horse serum albumin, dog serum albumin, rabbit serum albumin, rat serum albumin, or bovine (bovine/bovine subfamily) serum albumin. 30. The method of claim 29, wherein the broth, assay medium or solution is supplemented with human serum albumin, horse serum albumin, dog serum albumin or rabbit serum albumin. The method of any one of claims 29-31, wherein lysozyme is added to the broth, assay medium or solution. 32. The method of any one of claims 29-31, wherein human serum albumin is added to the broth, assay medium or solution at a concentration of 10% to 50% human serum albumin. 32. The method of any one of claims 29-31, wherein human serum albumin is added to the broth, assay medium or solution at a concentration of 20% to 40% human serum albumin. The method of any one of claims 29-31, wherein said antimicrobial peptide is a lysin polypeptide having an SH3 binding domain. 36. The method of claim 35, wherein said lysin polypeptide is selected from PlySs2 (CF-301) lysin, Sal lysin, LysK lysin, lysostaphin, fill lysin, LysH5 lysin, MV-L lysin, LysGH15 lysin and ALE-1 lysin. 36. The method of claim 35, wherein the lysin polypeptide is PlySs2 (CF-301), including the amino acid sequence presented in SEQ ID NO:3, or a variant thereof having at least 80% identity to the polypeptide of SEQ ID NO:3 and effective to kill Staphylococcus and Streptococcus bacteria. 32. The method of any one of claims 29-31, wherein a composition comprising an antimicrobial peptide that is a lysin polypeptide and further comprising one or more antimicrobial agents is evaluated. 39. The method of claim 38, wherein said one or more antimicrobial agents are antibiotics.

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